Dry powder treprostinil for the treatment of pulmonary hypertension

ABSTRACT

A dry powder inhalation treatment for pulmonary arterial hypertension includes a dose of dry particles comprising greater than 25 micrograms of treprostinil enclosed in a capsule. The dry particles can include treprostinil, a wetting agent, a hydrophobicity modifying agent, a pH modifying agent and a buffer. A method of treating a patient having pulmonary arterial hypertension includes providing a patient a dry powder inhaler, providing the patient at least one capsule for use in the dry powder inhaler, the capsule including at least 25 micrograms of treprostinil.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.17/104,348, filed Nov. 25, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/099,135 (now U.S. Pat. No. 10,898,494), filedMay 5, 2017, which is a U.S. national stage of International ApplicationNo. PCT/US2017/031301, filed May 5, 2017, which claims priority to andthe benefit of U.S. Provisional Patent Application No. 62/332,013, filedMay 5, 2016, U.S. Provisional Patent Application No. 62/404,960, filedOct. 6, 2016, U.S. Provisional Patent Application No. 62/440,078, filedDec. 29, 2016, and U.S. Provisional Patent Application No. 62/472,204,filed Mar. 16, 2017, all of which are incorporated herein by referencein their entireties.

TECHNICAL FIELD

The present invention provides an improvement to the treatment ofpulmonary hypertension, a condition that deteriorates the lives of manythousands of patients toward an untimely death. The present inventionprovides, for the first time, a stable, user friendly, uniform drypowder inhaled treprostinil formulation, methods of making, and usethereof in humans.

BACKGROUND

Pulmonary arterial hypertension (PAH) is a complex, multifactorial,progressive, and life-threatening disease characterized by proliferativeand obstructive changes in the pulmonary vasculature and involvingnumerous biochemical pathways and cell types. The disease ischaracterized by elevated pulmonary arterial pressure caused bynarrowing of the blood vessels in the lungs and, ultimately, rightventricular failure. The disease carries a poor prognosis associatedwith significant morbidity and mortality, having a historical survivalrate less than five years. PAH is a sub-group of pulmonary hypertension(PH), which is elevation of blood pressure in lungs. Endothelialdysfunction is thought to occur early on, leading to cell proliferationand structural changes in the pulmonary vasculature that lead toincreased pulmonary arterial pressure (PAP) and resultant rightventricular enlargement and dysfunction. In addition, endothelialdysfunction results in chronically impaired production of vasoactivemediators, such as nitric oxide (NO) and prostacyclin, along withprolonged overexpression of vasoconstrictors, such as endothelin-1.

PAH affects approximately 15 out of every one million individuals. Thereare approximately 1,000 new cases of PAH diagnosed in the United Stateseach year. The mean age at diagnosis is between 50 and 65 years of age,although the disorder may present much earlier in childhood or eveninfancy. While gender-based prevalence estimates for PAH are variable,estimates for the overall prevalence of pulmonary hypertension (PH) infemales is approximately twice that of males.

PAH is part of a larger classification for pulmonary hypertension whichis divided into five groups based on World Health Organization (WHO)criteria (designated as WHO Groups 1 through 5). PAH is used to describeexclusively WHO Group 1. Pulmonary hypertension is used to describe theremaining four groups (WHO Groups 2-5) and also when referring to all 5groups collectively.

-   -   WHO Group 1—PAH: Pulmonary arterial hypertension.    -   WHO Group 2—PH: Pulmonary hypertension secondary to left heart        disease.    -   WHO Group 3—PH: Pulmonary hypertension secondary to lung        diseases or hypoxemia.    -   WHO Group 4—PH: Chronic thromboembolic pulmonary hypertension.    -   WHO Group 5—PH: Pulmonary hypertension with unknown mechanisms.

PAH initially presents as exertional dyspnea, lethargy, and fatigue andis often confused for other disease states. As PAH progresses and rightventricular failure develops, exertional chest pain (i.e., angina),exertional syncope, and peripheral edema may develop. Followingconfirmation of diagnosis based on hemodynamic parameters, treatment isrecommended to lower pulmonary pressures and treat the symptoms of PAH.Although no cure exists for PAH, treatment of PAH is directed atimproving hemodynamic measures, New York Heart Association (NYHA)functional class, the 6 minute walk distance (6 MWD), quality of life,and, in some studies, survival.

The severity of PAH may be classified according to the NYHA heartfailure guidelines as follows:

-   -   NYHA Class I: Patients with no limitation of activities; they        suffer no symptoms from ordinary activities.    -   NYHA Class II: Patients with slight, mild limitation of        activity; they are comfortable with rest or with mild exertion.    -   NYHA Class III: Patients with marked limitation of activity;        they are only comfortable at rest.    -   NYHA Class IV: Patients who should be at complete rest, confined        to bed or chair; any physical activity brings on discomfort and        symptoms occur at rest.

While the exact underlying cause of PAH is unclear, mutations in thebone morphogenic protein receptor type II (BMPR2) gene account forapproximately 75% of familial PAH and up to 25% of apparently sporadicPAH cases. These mutations may promote cell division or prevent celldeath, resulting in an overgrowth of cells in smaller pulmonaryarteries. This overgrowth increases resistance to blood flow, triggeringhypertension. Additional genetic abnormalities may also contribute toPAH.

Currently Available Treatments

There are five classes of drugs that have been approved to treat PAH,including endothelin receptor antagonists (ERAs), phosphodiesterase type5 (PDE5) inhibitors, soluble guanylate cyclase stimulators, prostacyclinreceptor agonists, and prostacyclin analogs. Approved PAH therapies andtheir route of administration include:

-   -   ERA: bosentan (oral) and ambrisentan (oral)    -   PDE5: sildenafil (oral, intravenous (IV) and tadalafil (oral)    -   Soluble Guanylate Cyclase (sGC) Stimulator: riociguat (oral)    -   Prostacyclin Receptor Agonist: selexipag (oral)    -   Prostacyclin Analog: epoprostenol (IV) iloprost (inhaled), and        treprostinil (oral), (subcutaneous and IV), and (inhaled)

Treprostinil is a chemically stable tricyclic benzidine prostanoid withvasodilator properties that is capable of reducing pulmonaryvasoconstriction with minimal effects on systemic blood pressure.Treprostinil has been approved for the treatment of PAH under the tradenames REMODULIN® (United Therapeutics Corporation; subcutaneous or IVinfusion) and TYVASO® (United Therapeutics Corporation; inhaled viaultrasonic, pulsed nebulization delivery device). While both have proveneffective for PAH, one advantage of TYVASO's inhaled route ofadministration is that it brings the drug very near the desired site ofaction (pulmonary arteries in the lungs).

Despite the current treatment options for PAH patients, each optionincludes drawbacks, most notably for the inhaled route of administrationTyvaso requires use of a large, cumbersome nebulization device thatrequires power, water and user manipulation for cleaning and operating.Moreover, the nebulization device by its nature is not convenient to thepatient as compared to carrying a small, concealable dry powderinhalation device such as those used for treating asthma and many otherchronic and acute issues. Furthermore, nebulized treprostinil has shownclinical limitations on treprostinil dosing, which may limit theapplicability of the inhaled route of administration to a smallersubsector of PAH patients than necessarily treatable via the inhaledroute from a dry powder inhaled treprostinil product of the presentinvention.

SUMMARY OF THE INVENTION

The present inventors have developed and reduced to practice aninhalation dry powder formulation of treprostinil that is produced usingLiquidia's PRINT® Technology (Particle Replication in NonwettingTemplates), LIQUIDIA TECHNOLOGIES, INC. This PRINT particle formulationfor dry powder delivery of treprostinil (otherwise referred to asLIQ861) is under clinical evaluation. The present applicants intend touse the same indication (i.e., treatment of pulmonary arterialhypertension [WHO Group 1] in patients with NYHA Class III symptoms, toimprove exercise ability) dose and dose regimen (4×/day) as defined inthe approved nebulized treatment label (TYVASO® UNITED THERAPEUTICS). Inparticular, the present invention provides for dosing levels that exceedthe maximum tolerated dose delivered through a nebulizer. In some casesthe present invention may also treat other indications under thepulmonary hypertension disease states.

In some embodiments, a dry powder inhalation treatment for pulmonaryarterial hypertension according to the present invention includes a doseof dry particles comprising greater than 25 micrograms of treprostinilenclosed in a capsule. In some embodiments, the dose of dry particlescomprises from about 25 micrograms to about 400 micrograms oftreprostinil. In some embodiments, the dose of dry particles comprisesfrom about 50 micrograms to about 350 micrograms of treprostinil. Insome embodiments, the dose of dry particles comprises from about 75micrograms to about 300 micrograms of treprostinil. In some embodiments,the dose of dry particles comprises from about 100 micrograms to about300 micrograms of treprostinil. In some embodiments, the dose of dryparticles includes greater than or equal to 100 micrograms oftreprostinil. In some embodiments, the dose of dry particles comprisesgreater than or equal to 150 micrograms of treprostinil. In someembodiments, the dose of dry particles comprises greater than or equalto 200 micrograms of treprostinil. In some embodiments, the dose of dryparticles comprises greater than or equal to 250 micrograms oftreprostinil. In some embodiments, the dose of dry particles comprisesgreater than or equal to 300 micrograms of treprostinil. In someembodiments, the dose of dry particles includes greater than or equal to5 mg of the dry particles. In some embodiments, the dose of dryparticles includes greater than or equal to 10 mg of the dry particles.In yet other embodiments, the dose of dry particles includes greaterthan or equal to 15 mg of the dry particles. In further embodiments, adry powder treatment for pulmonary arterial hypertension, includes asingle capsule enclosing 5 mg or more dry particles comprising 25micrograms of treprostinil per each 5 mg of the dry particles.

In some embodiments, a method of treating a patient having pulmonaryarterial hypertension includes providing a patient a dry powder inhaler,providing the patient at least one capsule for use in the dry powderinhaler, wherein the capsule comprises at least 25 micrograms oftreprostinil, and instructing the patient to utilize the dry powderinhaler to inhale the treprostinil. In some such embodiments, thecapsule includes at least 50 micrograms of treprostinil. In someembodiments, the capsule includes at least 100 micrograms oftreprostinil. In some embodiments, the capsule comprises at least 150micrograms of treprostinil. In some embodiments, the capsule comprisesgreater than or equal to 200 micrograms of treprostinil. In someembodiments, the capsule comprises greater than or equal to 250micrograms of treprostinil. In some embodiments, the capsule comprisesgreater than or equal to 300 micrograms of treprostinil. In someembodiments, the capsule comprises from about 25 micrograms to about 400micrograms of treprostinil. In some embodiments, the capsule comprisesfrom about 50 micrograms to about 350 micrograms of treprostinil. Insome embodiments, the capsule comprises from about 75 micrograms toabout 300 micrograms of treprostinil. In some embodiments, the capsulecomprises from about 100 micrograms to about 300 micrograms oftreprostinil. In further embodiments, the patient may be prescribed touse two capsules per dose cycle per day, generally with PAH requiring 4times per day dosing. In some embodiments, the patient may be prescribedto use three capsules per day. In some embodiments, the patient may beprescribed to use four capsules per day. In some embodiments, a methodof treating a patient having pulmonary arterial hypertension includesdosing the patient having pulmonary arterial hypertension with a drypowder dose of treprostinil, wherein the dose of treprostinil is greaterthan 85 micrograms (e.g., about 100 micrograms to about 350 micrograms).In some embodiments, the patient may be dosed one, two, three, four, ormore times per day. A further method of treating a patient havingpulmonary arterial hypertension includes delivering, in dry powder,greater than 12.5 micrograms of treprostinil to a patient per breath. Inanother embodiment, a method of treating a patient having pulmonaryarterial hypertension includes delivering, in dry powder, greater than25 micrograms of treprostinil to a patient per breath. In anotherembodiment, a method of treating a patient having pulmonary arterialhypertension includes delivering, in dry powder, from about 12.5 toabout 50 micrograms of treprostinil to a patient per breath. In yetanother embodiment, a method of treating a patient having pulmonaryarterial hypertension includes delivering, in dry powder, about 25 toabout 50 micrograms of treprostinil to a patient per breath. In afurther embodiment, a method of treating a patient having pulmonaryarterial hypertension includes delivering, in dry powder, greater than50 micrograms of treprostinil to a patient per breath. In a furtherembodiment, a method of treating a patient having pulmonary arterialhypertension includes delivering, in dry powder, greater than or equalto 100 micrograms of treprostinil to a patient per breath. In a furtherembodiment, a method of treating a patient having pulmonary arterialhypertension includes delivering, in dry powder, greater than or equalto 150 micrograms of treprostinil to a patient per breath. In a furtherembodiment, a method of treating a patient having pulmonary arterialhypertension includes delivering, in dry powder, greater than or equalto 200 micrograms of treprostinil to a patient per breath.

A dry powder inhalation composition for treating pulmonary arterialhypertension according to a further embodiment includes a plurality ofdry powder particles comprising treprostinil, a non-reducing sugar, awetting agent, a hydrophobicity modifying agent, a pH modifying agentand a buffer. In some such embodiments, the bulking agent comprisestrehalose dihydrate. In some embodiments, the wetting agent comprisespolysorbate 80. In some embodiments, the hydrophobicity modifying agentcomprises L-leucine. In some embodiments, the pH modifying agentcomprises sodium citrate dihydrate. In some embodiments, the buffercomprises sodium chloride. In certain embodiments, the compositioncomprises less than about 4 percent by weight water. In someembodiments, the composition comprises less than about 2 percent byweight water. In some embodiments, the composition comprises less thanabout 1 percent by weight water.

In yet further embodiments, the dry powder particles include particleshaving a three dimensional shape including a width and length not lessthan 1 micrometer and not more than 2 micrometers and a depth not lessthan 0.3 micrometers and not more than 0.8 micrometers. In someembodiments, the dry powder particles comprise a dried solutioncomprising trehalose dihydrate, L-leucine, treprostinil sodium,polysorbate 80, sodium citrate dihydrate, sodium chloride and water. Insome embodiments, the dry powder particles comprise by percent solidsabout 0.581 percent treprostinil sodium, about 92.32 percent trehalose,about 2.19 percent polysorbate 80, about 4.39 percent L-leucine, about0.26 percent sodium citrate, and about 0.25 percent sodium chloride.

A method of making a particle for dry powder delivery to the lung of apatient in need thereof, in some embodiments, includes molding acomposition comprising about 12.30 weight percent trehalose dihydrate,about 0.53 weight percent L-leucine, about 0.07 weight percenttreprostinil sodium, about 0.26 weight percent polysorbate 80, about0.04 weight percent sodium citrate dihydrate, about 0.03 weight percentsodium chloride and about 86.78 weight percent water into a particle. Insome embodiments, the method of making the particle further includesdrying the composition such that the particle comprises less than 4percent by weight water.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention can beembodied in different forms and thus should not be construed as beinglimited to the illustrated embodiments set forth herein.

FIG. 1 shows a three-dimension rendering of a pollen particle accordingto an embodiment of the present invention.

FIG. 2 shows an example NGI distribution for active particles(PAH-1R-0943-010). For each of the three data sets represented for eachcollection cup, the beginning of the run is the left hand bar (A1), themiddle of the run is the center bar (B1), and the end of the run is theright hand bar (C1). Data was obtained using the Monodose Model 8 device(95 L/min, 2 sec).

FIGS. 3A and 3B are tables including data for Cohort 1 of a clinicaltrial. The table shown in FIG. 3A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 1. Preliminarynon-compartmental PK parameters for treprostinil are summarized in thetable shown in FIG. 3B.

FIGS. 4A and 4B are tables including data for Cohort 2 of a clinicaltrial. The table shown in FIG. 4A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 2. Preliminarynon-compartmental PK parameters for treprostinil for Cohort 2 aresummarized in the table shown in FIG. 4B.

FIGS. 5A and 5B are tables including data for Cohort 3 of a clinicaltrial. The table shown in FIG. 5A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 3. Preliminarynon-compartmental PK parameters for treprostinil for Cohort 3 aresummarized in the table shown in FIG. 5B.

FIGS. 6A and 6B are tables including data for Cohort 4 of a clinicaltrial. The table shown in FIG. 6A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 4. Preliminarynon-compartmental PK parameters for treprostinil for Cohort 4 aresummarized in FIG. 6B.

FIGS. 7A and 7B are tables including data for Cohort 5 for a clinicaltrial. The table shown in FIG. 7A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 5. Preliminarynon-compartmental PK parameters for treprostinil for Cohort 5 aresummarized in FIG. 7B.

FIGS. 8A, 8B, and 8C are tables including data for Cohort 6 for aclinical trial. The table shown in FIG. 8A contains a summary of thetreprostinil concentration-time data for individual subjects withdescriptive statistics for the six active subjects in Cohort 6-R.Preliminary non-compartmental PK parameters for treprostinil for Cohort6-R are summarized in FIG. 8B. Preliminary non-compartmental PKparameters for treprostinil for Cohort 6-Original are summarized in FIG.8C.

FIGS. 8D, 8E, 8F, and 8G contain data for the clinical trial. Meanconcentration-time data for each of the six cohorts is displayed on alinear scale in FIG. 8D. Plots of the relationship between dose and Cmaxand AUCinf are displayed in FIG. 8E and FIG. 8F, respectively. A plot ofthe relationship between dose and the oral clearance, CL/F, is shown inFIG. 8G.

FIG. 9 is an SEM image showing pollen-shaped particles according to anembodiment of the present invention.

FIG. 10 is a flow diagram showing a process of manufacturing particlesaccording to an embodiment of the present invention.

FIG. 11 shows an example dry powder inhalation device which may be usedto deliver particles to a patient in accordance with embodiments of thepresent invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Drug Substance

The drug substance (DS) according to embodiments of the presentinvention is treprostinil, which is a synthetic analog of prostacyclin(PGI₂). The IUPAC name for treprostinil is(2-[[(1R,2R,3aS,9aS)-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[g]naphthalen-5-yl]oxy]aceticacid).

Inhalation Powder Drug Product

The inhalation powder drug product according to certain aspects of thepresent invention provides a dry powder dosage form of treprostinil andexcipients formed into a particle (drug product intermediate, orDP-intermediate) that is, in some embodiments, filled into a capsule,for example, a hydroxypropyl methylcellulose (HPMC) capsule (size 3)(LIQ861). In some embodiments, the DP-intermediate is atreprostinil/excipient matrix from which particles of precise size andshape are formed according to the methods herein. In one example, theparticles of the DP-intermediate comprise a shape correspondinggenerally to a rounded triangular shape having a volume, where the innerportion of the rounded triangular shape, in size, fits a 1 micrometerequilateral triangle (otherwise referred to as being pollen-shaped). Athree-dimensional rendering of such a particle shape is depicted in FIG.1 . In another embodiment, the pollen-shape may be trefoil-shaped withan inscribed circle diameter of 1 micrometer, and a prescribed thicknessof a value or range between 0.5 and 1 micrometer, or more preferred 0.7micrometer. In addition, certain embodiments of the present drug productincludes particles having 0.5% treprostinil used in a first clinicalstudy to investigate dose levels of 25 mcg, 50 mcg, 75 mcg, 100 mcg, 125mcg and 150 mcg treprostinil in LIQ861. In further embodiments, a drugproduct according to the present invention may provide dose levels of175 mcg, 200 mcg, 225 mcg, 250 mcg, 275 mcg, 300 mcg, 325 mcg, or 350mcg treprostinil. In further embodiments, a drug product according tothe present invention may provide dose levels of 50 mcg treprostinilplus or minus 10 mcg, 9 mcg, 8 mcg, 7 mcg, 6 mcg, 5 mcg, 4 mcg, 3 mcg, 2mcg or 1 mcg treprostinil loaded into capsules for delivery to a patientin a dry powder. In further embodiments, a drug product according to thepresent invention may provide dose levels of 75 mcg treprostinil plus orminus 10 mcg, 9 mcg, 8 mcg, 7 mcg, 6 mcg, 5 mcg, 4 mcg, 3 mcg, 2 mcg or1 mcg treprostinil loaded into capsules for delivery to a patient in adry powder. In further embodiments, a drug product according to thepresent invention may provide dose levels of 100 mcg treprostinil plusor minus 10 mcg, 9 mcg, 8 mcg, 7 mcg, 6 mcg, 5 mcg, 4 mcg, 3 mcg, 2 mcgor 1 mcg treprostinil loaded into capsules for delivery to a patient ina dry powder. In further embodiments, a drug product according to thepresent invention may provide dose levels of 150 mcg treprostinil plusor minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% treprostinil loadedinto capsules for delivery to a patient in a dry powder. In furtherembodiments, a drug product according to the present invention mayprovide dose levels of 200 mcg treprostinil plus or minus 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2% or 1% mcg treprostinil loaded into capsules fordelivery to a patient in a dry powder. In further embodiments, a drugproduct according to the present invention may provide dose levels of300 mcg treprostinil plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%or 1% treprostinil loaded into capsules for delivery to a patient in adry powder.

According to the present invention, due to the formulation of thepresent dry powder particles, the particles remain stable for longperiods of time at relatively low humidity conditions. In someembodiments, the present invention provides dry powder particlespackaged under sealed conditions that remain stable for more than 3months at 40 degrees Celsius at 75 percent relative humidity. Therefore,the particles can be utilized to provide a patient with a dry powderinhaled drug form of treprostinil, not previously available until thepresent invention. This invention, in some embodiments, provides a userwith a reduction in interaction with drug product by removing therequirements on the patient to reconstitute their drug product for usein a nebulizer device. The patient is also enabled to receive equaldosing with more than 50 percent reduction in breath treatments on adevice, and in some embodiments more than 65 percent reduction in breathtreatments.

The present invention, in some embodiments, also provides a dryformulation of treprostinil, which upon delivery to a patient via theinhaled route, becomes soluble and pharmaceutically available in lessthan 10 seconds. In some embodiments, the dry formulation compositionbecomes soluble and pharmaceutically available in less than 5 seconds.In some embodiments, the dry formulation composition becomes soluble andpharmaceutically available in less than 2 seconds. In some embodiments,the dry formulation composition becomes soluble and pharmaceuticallyavailable in about 1 second. In some embodiments, the dry formulationcomposition becomes soluble and pharmaceutically available in less than1 second. In some embodiments, the dry formulation composition becomessoluble and pharmaceutically available in less than about 0.5 seconds.Furthermore, the excipients in the dry particle formulation of thepresent invention maintain pH and salt gradient during processing suchthat the active agent remains in a state to become soluble in the lungconditions of a user.

A detailed description of the LIQ861 formulation, particle composition,particle geometry, packaging, device, delivery, stability, dose, and adescription of the use follows.

In some embodiments, a formulation according to the present inventionincludes a drug substance (e.g., Treprostinil, Treprostinil Sodium)together with one or more excipients. In some embodiments, the one ormore excipients may include a bulking agent, a wetting agent, ahydrophobicity modifier, a pH modifier, a buffer component, orcombinations thereof. Examples of such formulations according to certainspecific embodiments are provided in the tables below.

LIQ861 Drug Product-Intermediate Description for Active (LIQ861) andPlacebo Formulations (Dihydrate Form Calculations)

Quantity (mg/g) Percent Component Function (Active) Solids TreprostinilSodium Drug Substance 5.3 0.53 (5.0 as treprostinil) Trehalose DihydrateBulking Agent 930 92.97 Polysorbate 80 Wetting Agent 20 2.00 L-LeucineHydrophobicity Modifier 40 4.00 Sodium Citrate pH Modifier 2.7 0.27Dihydrate Sodium Chloride Buffer Component 2.3 0.23LIQ861 Drug Product-Intermediate Description for Active (LIQ861) andPlacebo Formulations (Anhydrous Form Calculations)

Quantity (mg/g) Percent Normalized Component Function (Active) Solidsmg/g Treprostinil Sodium Drug Substance 5.3 0.581 5.81 (5.0 astreprostinil) Trehalose Bulking Agent 841 92.32 923.23 Polysorbate 80Wetting Agent 20 2.19 21.94 L-Leucine Hydrophobicity Modifier 40 4.3943.89 Sodium Citrate pH Modifier 2.4 0.26 2.60 Sodium Chloride BufferComponent 2.3 0.25 2.52

Inhalation Device

According to an embodiment of administering the present invention drugparticle, LIQ861 is administered using an RS00 Model 8 dry powderinhalation device (Plastiape S.p.A.). The present invention provides formulti-day administration of LIQ861 according to some embodiments.

Indication

The present invention, according to an embodiment, is useful for thetreatment of pulmonary arterial hypertension (WHO Group 1) in patientswith NYHA Class III symptoms, to improve exercise ability.

Chemistry, Manufacturing, and Controls (CMC)

Drug Substance (DS)

The drug substance according to embodiments of the present invention istreprostinil and the salt form used for LIQ861 is treprostinil sodium.Detailed information about treprostinil sodium, including physical andchemical properties, characterization, manufacturing and controls,container closure system, and stability attributes may be found in theDrug Master File (DMF) lodged with the FDA for treprostinil. Generalinformation on the DS is provided herein.

Nomenclature

The international non-proprietary name (INN) for LIQ861 is treprostinilsodium. The chemical name is2-((1R,2R,2R,3aS,9aS)-2-hydroxy-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalen-5-yloxy)aceticacid, sodium salt. The chemical abstracts service registration number is[289480-64-4].

Structure

The structure of treprostinil sodium is depicted herein below. Themolecular formula is C₂₃H₃₃NaO₅ and it has a molecular weight of 412.49daltons.

Chemical Structure of Treprostinil Sodium

General Properties

Treprostinil sodium appears as a white or pale yellowish powder. It isvery soluble in water and ethanol, very slightly soluble in acetone, andpractically insoluble in acetonitrile, n-hexane, and ethyl acetate. Thespecific optical rotation calculated with reference to the anhydrous andsolvent free basis is [α]_(D) ²⁰=+38.0°˜+44.0°. It is hygroscopic. ThepKa of treprostinil is 4.5, using aqueous titration with 20% ethanol asa co-solvent. The distribution coefficient of treprostinil in variousbuffer solutions at various pH levels indicates distribution intooctanol layers at all pH levels.

Inhalation Particle Drug Product—LIQ861

Description and Composition of the Drug Product Particle

The inhalation drug particle product, in some embodiments, includes orconsists of a dry powder dosage form of treprostinil and excipients(drug product-intermediate; DP-intermediate; or drug particle) that maybe filled into, for example, a HPMC capsule (size 3). TheDP-intermediate, in some embodiments, is a treprostinil/excipient matrixfrom which particles of precise size (e.g., 1 μm) and shape (e.g.,“pollen-shaped”) are created using Liquidia's PRINT Technology. The“pollen-shaped” particles may also be described as trefoil-shaped, withan inscribed circle diameter of 1 μm, and a thickness of 0.7 μm. Athree-dimensional rendering of such a particle shape is depicted in FIG.1 . LIQ861 comprised drug product capsule strengths of 25 mcg, 50 mcg,and 75 mcg treprostinil used in the first clinical study to investigateplanned dose levels of 25 mcg, 50 mcg, 75 mcg, 100 mcg, 125 mcg and 150mcg treprostinil. The 100 mcg, 125 mcg and 150 mcg doses may be made upof a combination of lower dose capsules. In further embodiments, a drugproduct according to the present invention may provide capsules withdose levels of 175 mcg, 200 mcg, 225 mcg, 250 mcg, 275 mcg, 300 mcg, 325mcg, or 350 mcg treprostinil. In further embodiments, a drug productaccording to the present invention may provide capsules with dose levelsof 50 mcg treprostinil plus or minus 10 mcg, 9 mcg, 8 mcg, 7 mcg, 6 mcg,5 mcg, 4 mcg, 3 mcg, 2 mcg or 1 mcg treprostinil for delivery to apatient in a dry powder. In further embodiments, a drug productaccording to the present invention may provide capsules with dose levelsof 75 mcg treprostinil plus or minus 10 mcg, 9 mcg, 8 mcg, 7 mcg, 6 mcg,5 mcg, 4 mcg, 3 mcg, 2 mcg or 1 mcg treprostinil for delivery to apatient in a dry powder. In further embodiments, a drug productaccording to the present invention may provide capsules with dose levelsof 100 mcg treprostinil plus or minus 10 mcg, 9 mcg, 8 mcg, 7 mcg, 6mcg, 5 mcg, 4 mcg, 3 mcg, 2 mcg or 1 mcg treprostinil for delivery to apatient in a dry powder. In further embodiments, a drug productaccording to the present invention may provide capsules with dose levelsof 150 mcg treprostinil plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2% or 1% treprostinil for delivery to a patient in a dry powder. Infurther embodiments, a drug product according to the present inventionmay provide capsules with dose levels of 200 mcg treprostinil plus orminus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% mcg treprostinil fordelivery to a patient in a dry powder. In further embodiments, a drugproduct according to the present invention may provide capsules withdose levels of 300 mcg treprostinil plus or minus 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2% or 1% treprostinil for delivery to a patient in a drypowder. A summary of the LIQ861 formulation, including powdercomposition, particle geometry, and a description of the dosing unitaccording to certain exemplary embodiments follows.

LIQ861 Drug Product-Intermediate Description for Active (LIQ861) andPlacebo Formulations (Dihydrate)

Quantity (mg/g) Percent Component Function (Active) Solids TreprostinilSodium Drug Substance 5.3 0.53 (5.0 as treprostinil) Trehalose DihydrateBulking Agent 930 92.97 Polysorbate 80 Wetting Agent/Process Aide 20 20L-Leucine Hydrophobicity Modifier 40 40 Sodium Citrate pH Modifier 2.70.27 Dihydrate Sodium Chloride Buffer Component 2.3 0.23Inhalation Drug Product Dosing Unit Description

Capsule Size 3 Opaque White HPMC Capsule Fill Description White toOff-White Powder Fill Particle Shape “pollen-shaped” Active Strength(μg) 0 (placebo)* 25 50 75 Formulation Powder 15 5 10 15 per Capsule(mg) *Excipients only (no treprostinil). Abbreviations: HPMC,hydroxypropyl methylcellulose

According to some embodiments of the present invention, drug particlesare provided that include a composition having a target dose of 15-90 μgof delivered treprostinil to the patient (current TYVASO® label is 18-54μg). In some embodiments of the present invention the dose oftreprostinil provided to the patient can be, for example, 100micrograms, 125 micrograms or 150 micrograms. In some embodiments of thepresent invention the dose of treprostinil provided to the patient, forexample, can contain about 100 micrograms, about 125 micrograms or about150 micrograms. In some embodiments, each dose contains greater than orequal to 200 micrograms of treprostinil. In some embodiments, each dosecontains greater than or equal to 225 micrograms of treprostinil. Insome embodiments, each dose contains greater than or equal to 250micrograms of treprostinil. In some embodiments, each dose containsgreater than or equal to 275 micrograms of treprostinil. In someembodiments, each dose contains greater than or equal to 300 microgramsof treprostinil. In some embodiments, each dose contains from about 10micrograms to about 15 micrograms, 15 micrograms to about 20 micrograms,20 micrograms to about 25 micrograms, 25 micrograms to about 30micrograms, about 30 micrograms to about 35 micrograms, about 35micrograms to about 40 micrograms, about 40 micrograms to about 45micrograms, about 45 micrograms to about 50 micrograms, about 50micrograms to about 55 micrograms, about 55 micrograms to about 60micrograms, about 60 micrograms to about 65 micrograms, about 65micrograms to about 70 micrograms, about 70 micrograms to about 75micrograms, about 75 micrograms to about 80 micrograms, about 80micrograms to about 85 micrograms, about 85 micrograms to about 90micrograms, about 90 micrograms to about 95 micrograms, about 95micrograms to about 100 micrograms, or about 100 micrograms to about 105micrograms of treprostinil. In some embodiments, each dose contains fromabout 100 micrograms to about 110 micrograms, 110 micrograms to about120 micrograms, 120 micrograms to about 130 micrograms, 130 microgramsto about 140 micrograms, about 140 micrograms to about 150 micrograms,about 150 micrograms to about 160 micrograms, about 160 micrograms toabout 170 micrograms, about 170 micrograms to about 180 micrograms,about 180 micrograms to about 190 micrograms, about 190 micrograms toabout 200 micrograms, about 200 micrograms to about 210 micrograms,about 210 micrograms to about 220 micrograms, about 220 micrograms toabout 230 micrograms, about 230 micrograms to about 240 micrograms,about 240 micrograms to about 250 micrograms, about 250 micrograms toabout 260 micrograms, about 260 micrograms to about 270 micrograms,about 270 micrograms to about 280 micrograms, about 280 micrograms toabout 290 micrograms, about 290 micrograms to about 300 micrograms,about 300 micrograms to about 310 micrograms, about 310 micrograms toabout 320 micrograms, about 320 micrograms to about 330 micrograms,about 330 micrograms to about 340 micrograms, or about 340 micrograms toabout 350 micrograms of treprostinil. In some embodiments, each dosecontains from about 25 micrograms to about 400 micrograms oftreprostinil. In some embodiments, each dose contains from about 25micrograms to about 350 micrograms of treprostinil. In some embodiments,each dose contains from about 25 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 50micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 100micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 125 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 150micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 175 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 200micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 225 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 250micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 275 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 50micrograms to about 75 micrograms of treprostinil. In some embodiments,each dose contains from about 50 micrograms to about 100 micrograms oftreprostinil. In some embodiments, each dose contains from about 50micrograms to about 150 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 100 micrograms oftreprostinil. In some embodiments, each dose contains from about 75micrograms to about 125 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 150 micrograms oftreprostinil. In some embodiments, each dose contains from about 75micrograms to about 175 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 200 micrograms oftreprostinil. In some embodiments, each dose contains from about 75micrograms to about 225 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 250 micrograms oftreprostinil. In some embodiments, each dose contains from about 75micrograms to about 275 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 75micrograms to about 325 micrograms of treprostinil. In some embodiments,each dose contains from about 75 micrograms to about 350 micrograms oftreprostinil. In some embodiments, each dose contains from about 100micrograms to about 125 micrograms of treprostinil. In some embodiments,each dose contains from about 100 micrograms to about 150 micrograms oftreprostinil. In some embodiments, each dose contains from about 100micrograms to about 175 micrograms of treprostinil. In some embodiments,each dose contains from about 100 micrograms to about 200 micrograms oftreprostinil. In some embodiments, each dose contains from about 100micrograms to about 225 micrograms of treprostinil. In some embodiments,each dose contains from about 100 micrograms to about 250 micrograms oftreprostinil. In some embodiments, each dose contains from about 100micrograms to about 275 micrograms of treprostinil. In some embodiments,each dose contains from about 100 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 100micrograms to about 325 micrograms of treprostinil. In some embodiments,each dose contains from about 100 micrograms to about 350 micrograms oftreprostinil. In some embodiments, each dose contains from about 125micrograms to about 150 micrograms of treprostinil. In some embodiments,each dose contains from about 125 micrograms to about 175 micrograms oftreprostinil. In some embodiments, each dose contains from about 125micrograms to about 200 micrograms of treprostinil. In some embodiments,each dose contains from about 125 micrograms to about 225 micrograms oftreprostinil. In some embodiments, each dose contains from about 125micrograms to about 250 micrograms of treprostinil. In some embodiments,each dose contains from about 125 micrograms to about 275 micrograms oftreprostinil. In some embodiments, each dose contains from about 125micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 125 micrograms to about 325 micrograms oftreprostinil. In some embodiments, each dose contains from about 125micrograms to about 350 micrograms of treprostinil. In some embodiments,each dose contains from about 150 micrograms to about 175 micrograms oftreprostinil. In some embodiments, each dose contains from about 150micrograms to about 200 micrograms of treprostinil. In some embodiments,each dose contains from about 150 micrograms to about 225 micrograms oftreprostinil. In some embodiments, each dose contains from about 150micrograms to about 250 micrograms of treprostinil. In some embodiments,each dose contains from about 150 micrograms to about 275 micrograms oftreprostinil. In some embodiments, each dose contains from about 150micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 150 micrograms to about 325 micrograms oftreprostinil. In some embodiments, each dose contains from about 150micrograms to about 350 micrograms of treprostinil. In some embodiments,each dose contains from about 175 micrograms to about 200 micrograms oftreprostinil. In some embodiments, each dose contains from about 175micrograms to about 225 micrograms of treprostinil. In some embodiments,each dose contains from about 175 micrograms to about 250 micrograms oftreprostinil. In some embodiments, each dose contains from about 175micrograms to about 275 micrograms of treprostinil. In some embodiments,each dose contains from about 175 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 175micrograms to about 325 micrograms of treprostinil. In some embodiments,each dose contains from about 175 micrograms to about 350 micrograms oftreprostinil. In some embodiments, each dose contains from about 200micrograms to about 225 micrograms of treprostinil. In some embodiments,each dose contains from about 200 micrograms to about 250 micrograms oftreprostinil. In some embodiments, each dose contains from about 200micrograms to about 275 micrograms of treprostinil. In some embodiments,each dose contains from about 200 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 200micrograms to about 325 micrograms of treprostinil. In some embodiments,each dose contains from about 200 micrograms to about 350 micrograms oftreprostinil. In some embodiments, each dose contains from about 225micrograms to about 250 micrograms of treprostinil. In some embodiments,each dose contains from about 225 micrograms to about 275 micrograms oftreprostinil. In some embodiments, each dose contains from about 225micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 225 micrograms to about 325 micrograms oftreprostinil. In some embodiments, each dose contains from about 225micrograms to about 350 micrograms of treprostinil. In some embodiments,each dose contains from about 250 micrograms to about 275 micrograms oftreprostinil. In some embodiments, each dose contains from about 250micrograms to about 300 micrograms of treprostinil. In some embodiments,each dose contains from about 250 micrograms to about 325 micrograms oftreprostinil. In some embodiments, each dose contains from about 250micrograms to about 350 micrograms of treprostinil. In some embodiments,each dose contains from about 275 micrograms to about 300 micrograms oftreprostinil. In some embodiments, each dose contains from about 275micrograms to about 325 micrograms of treprostinil. In some embodiments,each dose contains from about 275 micrograms to about 350 micrograms oftreprostinil. In some embodiments, each dose contains from about 300micrograms to about 325 micrograms of treprostinil. In some embodiments,each dose contains from about 300 micrograms to about 350 micrograms oftreprostinil. In some embodiments, each dose contains from about 325micrograms to about 350 micrograms of treprostinil. In some embodiments,each dose contains from about 350 micrograms to about 375 micrograms oftreprostinil. In some embodiments, each dose contains from about 375micrograms to about 400 micrograms of treprostinil. In some embodiments,a patient may be provided with one, two, three, four, or more doses perday. In some embodiments, a patient may be provided up to one, two,three, or four doses per day. Each dose may be contained in a singlecapsule according to some embodiments, for example, a HPMC capsule (size3). In other embodiments, a dose may be made up of a combination oflower dose capsules. In some embodiments, a patient may be provided withfour doses per day to match the current treatment cycle (nebulizedtreprostinil) however the drug dose per treatment cycle under thepresent invention dry powder provides significantly higher dose levelsto be safely administered, such as for example, up to 100 mcg oftreprostinil per dosing, up to 125 mcg of treprostinil and up to 150 mcgof treprostinil per dosing as each were surprisingly demonstrated in thefirst clinical trial of LIQ861. In alternative embodiments, a patientmay be provided with four doses per day to match the current treatmentcycle (nebulized treprostinil) however the drug dose per treatment cycleunder the present invention dry powder provides significantly higherdose levels to be achieved, such as for example, up to 200 mcg oftreprostinil per dosing and up to 300 mcg of treprostinil per dosing assurprisingly demonstrated in pre-clinical toxicology studies usingLIQ861.

Treprostinil itself is poorly soluble in unbuffered water and low pHbuffers. However, the solubility improves with increasing pH as thecarboxylic acid is deprotonated. The sodium salt was selected for use inthis product since it enhances dissolution in aqueous media andfacilitates processing.

Excipients

According to some embodiments of the present invention, theDP-intermediate (anhydrous) is comprised of particles that include, forexample, the following excipients: trehalose, polysorbate 80, L-leucine,sodium citrate, and sodium chloride. In some embodiments, the ratio oftreprostinil sodium and excipients is 0.581:92.32:2.19:4.39:0.26:0.25(wt:wt solids) treprostinil sodium:trehalose:polysorbate80:leucine:sodium citrate:sodium chloride. A summary of the function,quantity, and compendial status of these excipients is provided herein.

The excipients were selected based upon the following functionalrequirements for the formulation:

-   -   Trehalose Dihydrate: Trehalose comprises the bulk of the        particle and was selected because it is a non-reducing sugar        with a high glass transition temperature. Trehalose is an        example of a non-reducing sugar (as opposed to lactose, which is        a reducing sugar) that can be used in the present invention.        Trehalose is more chemically compatible with compounds        containing primary amines, such as leucine.    -   Ultra-Pure Polysorbate 80 (Ultra-Pure Tween 80): Polysorbate 80        is added as a processing aide/wetting agent to facilitate        particle manufacturing. In some embodiments, Polysorbate 80 is a        particle processing aide and enables film generation during        particle manufacture by decreasing dewetting, leading to uniform        particle morphology.    -   L-leucine: Leucine is added as a hydrophobicity and surface        modifier to reduce the hygroscopicity of the particle and        improve aerosol efficiency. L-leucine is an example of a        formulation additive to reduce hygroscopicity to improve        stability of the final drug product powder.    -   Sodium chloride and sodium citrate: Sodium citrate and sodium        chloride are used to buffer the stock solution used in the PRINT        Technology manufacturing process and to help control acidity in        the particle. Sodium chloride and sodium citrate are examples of        buffers that help maintain pH and control ionization/acidity of        the formulation. In some embodiments of the present invention,        pH is maintained between about pH 6.0 and 7.2.

In addition to the active pharmaceutical ingredient the present drugparticle comprises a bulking agent, wetting agent, hydrophobicitymodifier, pH modifier and buffer. In some embodiments, the present drugparticle comprises, along with the active ingredients, a bulking agent,hydrophobicity controlling agent, and a pH controlling agent.

According to another embodiment of the present invention, LIQ861contains five excipients as follows: treprostinil sodium:trehalosedihydrate:leucine:polysorbate 80:sodium citrate dihydrate:sodiumchloride at ratios of 0.53:92.97:4:2:0.27:0.23. At an exampletreprostinil dose level of 100 μg/day of the present invention drugparticles, a patient would receive the following daily excipient doses:

-   -   18.6 mg of trehalose dihydrate. Assuming a patient weighs 60 kg        and has a lung mass of 1000 g, this is equivalent to 310 μg/kg        and 18.6 μg/g of lung.    -   0.4 mg of polysorbate 80. Assuming a patient weighs 60 kg and        has a lung mass of 1000 g, this is equivalent to 6.7 μg/kg and        0.4 μg/g of lung.    -   0.8 mg of leucine. Assuming a patient weighs 60 kg and has a        lung mass of 1000 g, this is equivalent to 13.3 μg/kg and 0.8        μg/g of lung.    -   0.05 mg of sodium citrate and 0.05 mg of sodium chloride.        Assuming a patient weighs 60 kg and has a lung mass of 1000 g,        this is equivalent to 0.83 μg/kg for each compound and 0.05 μg/g        of lung for each compound.

Formulation Development

According to embodiments of the present invention, LIQ861 has beendeveloped as a novel formulation of treprostinil for the treatment ofPAH. Treprostinil is currently approved for use in the treatment of PAHby subcutaneous, IV, oral, and inhalation routes of administration.TYVASO is currently the only marketed inhaled formulation oftreprostinil and is formulated as a liquid solution for administrationusing a nebulizer. The nebulized treprostinil is dosed, at maintenancedose, of 6 mcg drug per breath over 9 breaths for a dose of 54 mcg perdosing session. The nebulized treprostinil also has a maximum tolerateddose of 84 mcg over a dosing session with 14 breaths.

LIQ861 is suitable for inhaled administration using a dry powderinhalation device. The physicochemical properties and performancecharacteristics, manufacturing process and packaging, and stabilitycharacteristics of the DP have been studied, and a suitable formulationhas been identified for progression into human studies.

Physiochemical and Biological Properties

The “pollen-shaped” LIQ861 particles according to certain embodimentshave an aerodynamic size to enable efficient delivery to the pulmonaryarterioles (1≤MMAD≤5 μm) with a high FPF to limit oropharyngealdeposition. A scanning electron microscopy (SEM) image of the“pollen-shaped” feature is provided in FIG. 9 . The formulation ofexample particles shown in FIG. 9 is:treprostinil:trehalose:leucine:polysorbate 80:sodium citrate:sodiumchloride (Batch LKI-1R-983-27). Example aerosol data for the activeparticles are also provided in the table below.

During the development of the LIQ861 formulation, the applicants testedother possible particle shapes and sizes (e.g., 1.5 μm donut, 3.0 μmdonut). Based upon these studies, the applicants observed that the“pollen-shaped” feature resulted in a greater FPF, reduced MMAD,acceptable ED, and dose uniformity characteristics when compared toother features both with and without treprostinil.

Representative Aerosol Data (NGI) for Active Particles

MMAD ED FPF Sample (μm) GSD (% nominal) (% ED) Treprostinil Sodium: 1.881.99 64 83 Trehalose:Leucine:Polysorbate 80:Sodium Citrate:SodiumChloride (“pollen-shaped”) Abbreviations: NGI, Next GenerationImpactor ™, MSP Corp.; MMAD, mass median aerodynamic diameter; GSD,geometric standard deviation; ED, emitted dose; FPF, fine particlefraction; wt, weight. Batch LKI-1R-0983-21.

Manufacture

The manufacturing of LIQ861 particles according to some embodiments ofthe present invention is described below. A process flow diagram for theparticles (also referred to as DP-intermediate) according to someembodiments is shown in FIG. 10 .

In particular embodiments, the particles of the present disclosure arefabricated using PRINT® Technology (Liquidia Technologies, Inc.,Morrisville, N.C.) particle fabrication. In particular, the particlesare made by molding the materials intended to make up the particles inmold cavities.

In some embodiments, the molds can be polymer-based molds and the moldcavities can be formed into any desired shape and dimension. Uniquely,as the particles are formed in the cavities of the mold, the particlesare highly uniform with respect to shape, size, and composition. Due tothe consistency among the physical and compositional makeup of theparticles of the present compositions, the compositions of the presentdisclosure provide highly uniform release rates and dosing ranges.Methods and materials that may be used for fabricating the particlesaccording to embodiments of the present disclosure are further describedand disclosed in issued patents and patent applications, each of whichare incorporated herein by reference in its entirety: U.S. Pat. Nos.8,518,316; 8,444,907; 8,420,124; 8,268,446; 8,263,129; 8,158,728;8,128,393; 7,976,759; U.S. patent application publications Nos.2013-0249138, 2013-0241107, 2013-0228950, 2013-0202729, 2013-0011618,2013-0256354, 2012-0189728, 2010-0003291, 2009-0165320, 2008-0131692;and pending U.S. application Ser. No. 13/852,683 filed Mar. 28, 2013 andSer. No. 13/950,447 filed Jul. 25, 2013.

Particle Fabrication

An aqueous stock solution is prepared at the desired total solidsconcentration. All other excipients are combined with treprostinil andthen filtered prior to particle fabrication.

The stock solution is applied in a thin layer to a continuouspolyethylene terephthalate (PET) substrate backing layer. Forced airheat is used to drive off the water resulting in a dry film oftreprostinil and excipients. The dried film is then brought into contactwith a mold film, having cavities of the desired shape and size whichthe drug product particles will mimic, at an elevated temperature. Thedrug/excipient blend flows into the cavities of the mold, conforming tothe shape defined by the cavity. The result is a uniform array ofparticles adhered to a PET backing layer. The particles are then allowedto cool to room temperature as the roll is wound up for latercollection.

In one example of the present drug particles, the following stocksolution is used:

Stock Solution Components Used for Manufacture of TreprostinilParticles, According to an Embodiment:

Target Solution Target Solution Concentration Concentration Stockcomponent (Active) (Placebo) Target Trehalose   12% 12.7% Adjusted basedon mass balance of other formulation components Leucine 0.52% 0.54%0.52-0.54% (4% solids) Treprostinil Sodium 0.069%     0% 0.069% (0.53%solids) Polysorbate 80 0.26% 0.27% 0.26-0.28% (2% solids) Sodium Citrate0.035%  0.037%  Maintain pH stock solution for stability of treprostinilNaCl 0.030%  0.031%  Maintain tonicity of stock solution Diluent (water)87.0% 86.4% 86-91% evaluated; to coat appropriate formulation mass forprocessing and solubility of excipient component(s)

Dry Collection and Drying

Next, the particles are dry collected, the process of removing themolded particles from the PET backing layer and thereby creating a bulkpowder. The mold is first separated from the PET backing layer, exposingthe particle array attached to the PET backing layer. The particle arrayis then passed across a blade, in some embodiments a plastic blade, todislodge the particles from the backing layer. The particles can then becollected into a bulk powder for further processing.

Humidity is controlled to less than 15% RH during collection, in someembodiments due to the hygroscopicity of the powder. Temperature ismaintained at ambient, typically between 15 and 25° C.

Drying and Bulk Packaging

The drug particles are dried at less than or equal to 150 mTorr ofnitrogen or dry air for at least 2 days in a benchtop lyophilizer atroom temperature, according to some embodiments.

In some embodiments, the particles of the present invention are dried toless than about 10 percent water content. In some embodiments, theparticles of the present invention are dried to less than about 5percent water content. In further embodiments, the particles of thepresent invention are dried to less than about 4 percent water content.In still further embodiments, the particles of the present invention aredried to less than about 2 percent water content. In a preferredembodiment, the product is dried to less than about 1 percent watercontent by Karl Fisher titration.

Batch-to-Batch Uniformity of Drug Particles

In some embodiments, the particle uniformity from batch-to-batchprovides the present invention with an unexpected and exceptionaladvantage over the prior art. In certain embodiments, the uniformitywithin any given batch is unexpected and exceptionally advantageous overthe prior art. The present invention includes highly conserved batchuniformity as shown in the following data. See the table below and alsoFIG. 2 .

-   -   Uniformity: Sample aerosol data (NGI) for active particles        (PAH-1R-0974-010)

ED FPF F345 Sample MMAD GSD (% rec) (% ED) (fill) PAH-1R-0974-010-A1First 1.74 1.88 81% 88% 42% PAH-1R-0974-010-B1 Middle 1.80 1.87 78% 87%44% PAH-1R-0974-010-C1 Last 1.72 1.87 90% 89% 40%In the example shown, fine particle fraction remained within plus/minus1 percent within a single batch run.

Capsule Filling and Packaging

In some embodiments, HPMC capsules are filled with the DP-intermediatein a humidity controlled ISO 8 environment using an XCELODOSE®(Capsugel) instrument. The filled HPMC capsules are packaged in a lowhumidity environment. Ten capsules are placed in a DESICAP® Vial andclosed with a DESICAP® Cap. The closed vial is then placed into a foilbag with a desiccant canister prior to heat sealing the foil bag to formthe packaged drug product.

Stability Studies

According to the formulation of the present drug particle, it wasdesired to minimize uncontrolled exposure to ambient humidity. The drugparticles according to embodiments of the present invention are shown tobe stable for at least 9 months when stored under controlled humidityconditions at 25° C./60% RH. In some embodiments, the drug particles areshown to be stable for at least 6 months when stored under controlledhumidity conditions at 40° C./75% RH. In some embodiments, the drugparticles are shown to be stable for at least 9 months when stored underdesiccated conditions at 25° C./60% RH. In some embodiments, the drugparticles are shown to be stable for at least 6 months when stored underdesiccated conditions at 40° C./75% RH. Studies were conducted todetermine the stability of the drug particles at 25° C./60% RH and 40°C./75% RH.

Prototype Stability Study

The purpose of the Prototype Stability Study was to evaluate thestability of drug particles in capsules. Both the 25 and 75 μg strengthswere evaluated when stored at 25° C./60% RH and 40° C./75% RH. For thestudy, drug particles were placed into size 3 HPMC opaque capsules(Capsugel Vcaps). Ten filled capsules were placed into HDPE vials(Desicap) which were sealed with a stopper. The stoppered vial wasplaced into a foil overwrap with desiccant sachets.

Data for the 25 μg dose drug particles stored at 25° C./60% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months 9Months Assay 0.450-0.550% w/w 0.489 0.520 0.493 0.494 0.486 Treprostinilas free acid (%) Aerodynamic Report Results MMAD 1.92 2.11 2.2 2.1 2.1Particle Size (μm) 1.72 1.67 1.6 1.6 1.7 Distribution GSD 87 84 82.983.6 83.6 (μm) FPF (%) Delivered Report Results Average 19.9 21.6 19.6519.47 18.86 Dose (μg) Uniformity

Data for the 25 μg dose drug particles stored at 40° C./75% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.489 0.512 0.502 0.492 Treprostinil as free acid (%)Aerodynamic Report MMAD 1.92 2.09 2.1 2.1 Particle Size Results (μm)1.72 1.65 1.6 1.6 Distribution GSD (μm) 87 85 84.8 84.7 FPF (%)Delivered Dose Report Average 19.9 21.2 18.86 19.28 Uniformity Results(μg)

Data for the 75 μg dose drug particles stored at 25° C./60% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months 9Months Assay 0.450-0.550% w/w 0.489 0.500 0.496 0.494 0.487 Treprostinilas free acid (%) Aerodynamic Report MMAD 2.13 2.17 2.2 2.2 2.2 ParticleSize Results (μm) 1.60 1.61 1.6 1.6 1.6 Distribution GSD (μm) 85 84 84.283.7 82.3 FPF (%) Delivered Dose Report Average 63.6 63.0 60.76 59.6260.01 Uniformity Results (μg)

Data for the 75 μg dose drug particles stored at 40° C./75% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.489 0.509 0.506 0.491 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.13 2.14 2.2 2.1 Particle Size Results GSD(μm) 1.60 1.61 1.6 1.6 Distribution FPF (%) 85 85 84.8 85.3 DeliveredDose Report Average (μg) 63.6 61.0 59.86 59.42 Uniformity Results

Clinical Trial Material Stability Study

The purpose of the Clinical Trial Material Stability Study was toevaluate the stability of drug particles in capsules. Three strengthswere evaluated: 25, 50, and 75 μg active agent doses within capsules. Asin the previous study, two storage conditions were evaluated: 25° C./60%RH and 40° C./75% RH. For the study, drug particles were placed intosize 3 HPMC opaque capsules (Capsugel Vcaps). Ten filled capsules wereplaced into HDPE vials (DESICAP) which were sealed with a stopper. Thestoppered vial was placed into a foil overwrap with desiccant sachets.

Data for the 25 μg dose drug particles stored at 25° C./60% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.520 0.505 0.504 0.504 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.3 2.3 2.2 2.1 Particle Size Results GSD(μm) 1.6 1.6 1.6 1.6 Distribution FPF (%) 84.1 82.7 83.6 85.0 DeliveredDose Report Average (μg) 19.784 20.48 19.24 19.47 Uniformity Results(μg)

Data for the 25 μg dose drug particles stored at 40° C./75% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.520 0.518 0.501 0.506 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.3 2.2 2.2 2.2 Particle Size Results GSD(μm) 1.6 1.6 1.6 1.6 Distribution FPF (%) 84.1 84.1 85.8 84.7 DeliveredDose Report Average (μg) 19.784 20.73 18.89 18.80 Uniformity Results

Data for the 50 μg dose drug particles stored at 25° C./60% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.515 0.509 0.509 0.506 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.2 2.2 2.2 2.2 Particle Size Results GSD(μm) 1.6 1.6 1.6 1.6 Distribution FPF (%) 86.2 85.6 85.7 85.3 DeliveredDose Report Average (μg) 40.417 40.75 39.14 40.05 Uniformity Results(μg)

Data for the 50 μg dose drug particles stored at 40° C./75% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.515 0.520 0.505 0.501 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.2 2.2 2.2 2.2 Particle Size Results GSD(μm) 1.6 1.6 1.6 1.6 Distribution FPF (%) 86.2 86.5 86.2 84.1 DeliveredDose Report Average (μg) 40.417 39.55 38.96 37.50 Uniformity Results

Data for the 75 μg dose drug particles stored at 25° C./60% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.517 0.512 0.509 0.508 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.3 2.3 2.3 2.2 Particle Size Results GSD(μm) 1.6 1.6 1.6 1.6 Distribution FPF (%) 84.8 84.1 84.5 85.1 DeliveredDose Report Average (μg) 61.851 63.91 59.88 60.25 Uniformity Results(μg)

Data for the 75 μg dose drug particles stored at 40° C./75% RH is shownin the table below.

Time Points Test Specifications Initial 1 Month 3 Months 6 Months Assay0.450-0.550% w/w 0.517 0.513 0.503 0.495 Treprostinil as free acid (%)Aerodynamic Report MMAD (μm) 2.3 2.3 2.2 2.2 Particle Size Results GSD(μm) 1.6 1.6 1.6 1.6 Distribution FPF (%) 84.8 85.1 85.8 84.9 DeliveredDose Report Average (μg) 61.851 61.94 58.61 58.17 Uniformity Results

Dry Powder Inhalation Device

The RS00 Model 8 is a commercially available monodose dry powderinhalation device that is manufactured by Plastiape S.p.A (Italy) inaccordance with ISO and FDA standards. The overall design of RS00 Model8 device is shown in FIG. 11 .

A cap, which is retained on the mouthpiece, is designed to preventingress of dirt and other foreign material into the inhaler when not inuse. The plastic side portions cover the air inlet holes, but the capdoes not provide a hermetic seal to the device. The cap does not formpart of the actuation process.

When assembled, the mouthpiece is mounted on the inhaler body, but isremovable for cleaning purposes. To assemble the mouthpiece, the off-setpeg at the base of the mouthpiece is placed into the corresponding holein the inhaler body and the mouthpiece is rotated until it snaps closed.The snap closure ensures that the mouthpiece and inhaler body areproperly aligned and that no spurious airflow occurs. The mouthpiececontains a mesh that aids particle size reduction and prevent capsuleingestion during inhalation.

The inhaler body component contains two side buttons, each housing fourpins for piercing a capsule. The pins are inserted in the correspondinghousing of the pushbuttons and the heads of the pins are retained intheir position by a back-plate that is ultrasonically welded to thepushbutton. The buttons and pins are each maintained in their outwardposition by four small steel springs in each button. A three-componentsnap-lock system on the inhaler body ensures correct alignment of themouthpiece when closed.

A capsule piercing area is located internally, adjacent to the pins.When a capsule is inserted in this area, depressing the buttons causesthe button pins to pierce the capsule ends, thereby preparing thecapsule for emptying. Above the capsule piercing area, there are 2tangential air inlets and a circular chamber. These allow the capsule tospin when the patient inhales through the device. Capsule spinningcreates a centrifugal effect on the powder that promotes efficientemptying.

The performance of the premetered dry powder inhaler is a combination ofthe characteristics of LIQ861 (including the powder and capsule) and theinhalation device itself.

Nonclinical Studies

Treprostinil is a tricyclic benzidine analogue of endogenous PGI₂. Themajor pharmacologic actions of treprostinil are direct vasodilation ofpulmonary and systemic arterial vascular beds and inhibition of plateletaggregation. It was developed for chronic administration as a continuoussubcutaneous infusion as a treatment for patients with PAH. PGI₂, anendothelial cell derived substance, is a potent vasodilator andinhibitor of platelet aggregation. The hemodynamic properties oftreprostinil are similar to those of PGI₂ but, unlike PGI₂, treprostinilis chemically stable.

A series of in vivo studies were conducted to evaluate pharmokinetics(PK) and toxicology of the present invention dry powder treprostinilformulation.

Pilot, Non-GLP, Single-Dose, Inhalation PK Study of Treprostinil in Dogs(Study 19073)

This study compared the single dose PK of LIQ861 (administered via DPI)to Tyvaso (administered via nebulizer) at a target lung deposition of 3μg/kg in 3 beagle dogs. Results showed generally similar treprostinil PKprofiles following dosing with LIQ861 compared with Tyvaso. In thispilot single administration PK study, treprostinil (dry powderformulation; estimated lung deposition of 3.0 to 3.4 μg/kg) andtreprostinil (nebulized liquid; target lung deposition of 3 μg/kg) werecompared in 3 beagle dogs. The results showed generally similartreprostinil PK profiles following dosing with treprostinil (dry powderformulation) compared with treprostinil (nebulized liquid). The studydesign and results are discussed in more detail herein.

The applicants conducted a study comparing plasma concentrations andpharmacodynamics (PD) following administration of treprostinil sodium(nebulized liquid versus a dry powder formulation similar to the LIQ861formulation of the present invention) as a single inhalation exposure(via controlled ventilation) to anesthetized beagle dogs. Treprostinilsodium was prepared as a nebulized liquid from the same DS used toprepare the dry powder formulation. The dry powder formulation wasmanufactured using PRINT Technology and utilized the same drugsubstance, treprostinil sodium, but was different in excipientconcentrations compared to LIQ861. Importantly, the excipientconcentrations of the present invention provide highly consistent andreproducible batch to batch manufacturing of the LIQ861 product. Theformulations used in this study will be referred to as treprostinil(nebulized liquid) and treprostinil (dry powder formulation),respectively, in description of this study. The study design, results,and conclusions are described below.

In study 19073, 3 dogs received a single inhalation administration ofnebulized treprostinil (nebulized liquid; estimated lung deposition of3.4 μg/kg). After a 2-day washout, the dogs received a single inhalationadministration of treprostinil (dry powder formulation; estimated lungdeposition of 3.0 to 3.4 μg/kg). Blood was collected for plasma analysisof treprostinil concentrations prior to each administration and at 2, 5,10, 20, 30, 60, 120, and 180 minutes after the completion of eachadministration. In addition, 2 different dogs (one assigned to eachtreprostinil formulation) were used to monitor the following PDendpoints (hemodynamic changes): systemic arterial blood pressures [meanarterial pressure (MAP, mmHg), systolic arterial pressure (mmHg),diastolic arterial pressure (mmHg)], pulmonary artery pressure (PAP,mmHg), right atrial pressure (RAP, mmHg), pulmonary capillary wedgepressure (PCWP, mmHg) or left atrial pressure (mmHg), cardiac output(CO, L/min the average of 3), total peripheral resistance (TPR),pulmonary vascular resistance (PVR), and heart rate (HR). The PD effectswere assessed prior to initiation of dose administration and at targettimes of 5, 10, 20, 30, 60, 120, and 180 minutes after the completion ofthe administration. In contrast to the first three dogs, the dogsassigned to monitor the PD effects were anesthetized for the duration ofdata collection. The dog assigned treprostinil (nebulized liquid)received an estimated lung deposition of 4.0 μg/kg and the dog assignedto treprostinil (dry powder formulation) received an estimated lungdeposition of 2.5 μg/kg. Blood was collected at the same time points asfirst 3 dogs.

Treprostinil (nebulized liquid and dry powder formulation) had no effecton HR, PAP, RAP, PCWP, or CO, but had a slight effect on decreasing andthen increasing arterial blood pressure. Treprostinil (nebulized liquid)appeared to decrease stroke volume, increase TPR, and decrease PVR.Treprostinil (dry powder formulation) appeared to increase strokevolume, decrease TPR, and decrease PVR. The Study Director concludedthat the pilot data were inconclusive for comparing the potential PDeffects of treprostinil (nebulized liquid) to the treprostinil (drypowder formulation) formulation; however, there appeared to be noimportant differences in PD effects associated with administration oftreprostinil in either formulation.

Pilot, Non-GLP, Single-Dose, Inhalation PK Study of LIQ861 in Male Rats(Study 75670)

This study evaluated the PK of treprostinil in male rats followingsingle inhalation of a range of LIQ861 doses up to a feasible dose.Systemic exposure data from this study was used to determine appropriatedoses and blood sampling times for a definitive, comparative PK bridgingstudy of LIQ861 and nebulized treprostinil. Results from this study wereused to select dose levels and an optimal blood sampling paradigm for adefinitive PK bridging study.

Summary: Study 75670

The objective of the study was to determine the pharmacokinetic profileof treprostinil in male Sprague Dawley rats when administered as thetest item, PRINT Treprostinil dry powder (PRINT-Tre), as a single 4 hourinhalation at targeted dose levels of 0.15, 0.75, and 1.5 mg/kg. Resultsfrom this study will be used to determine appropriate dose levels andsampling time points for a definitive PK bridging study.

The test item was administered once by inhalation to 3 male rats pergroup as described in the table below:

Achieved Mean Total Inhaled Dose Achieved Aerosol Achieved Aerosol Levelof Treprostinil Concentration of Concentration of Group No. GroupDesignation (mg/kg/day) Treprostinil (μg/L) Trehalose (μg/L) 1 Low Dose0.158 1.06 150.46 2 Mid Dose 0.707 4.72 664.85 3 High Dose 1.409 9.391298.81

Assessments of mortality, clinical signs and body weights wereperformed. Blood samples were collected and analyzed for treprostinilcontent.

No mortality occurred. No clinical signs were observed and body weightswere unaffected.

The overall achieved gravimetric and analytical aerosol concentrationsfor all groups were within 16% of the targeted concentrations.Corresponding average treprostinil dose levels for all groups werewithin 7% of the targeted dose levels and a clear dose differentiationbetween groups for each sex was achieved. The gravimetric particle sizeMMADs from all groups were between 1.2 and 1.6 μm (GSD 2.06 to 2.56).For both treprostinil and trehalose, the chemical determination ofparticle size distribution ranged from 1.3 to 1.8 μm with thecorresponding GSDs between 1.65 and 2.15. The particle size distributionwas considered respirable gravimetrically and chemically.

Mean PK parameters for PRINT-Tre treatment groups obtained bynon-compartmental analysis of the mean treprostinil plasma concentrationdata sets are summarized as follows:

T_(1/2) T_(max) C_(max) AUC_(0-Tlast) AUC_(INF) Group (hr) (hr) (ng/mL)(hr*ng/mL) (hr*ng/mL) 1 Mean 1.01 3.75 6.800 17.320 18.335 SD 0.521 0.000.951 2.281 1.806 N 3 3 3 3 3 2 Mean 1.68 3.75 31.933 81.289 93.369 SD0.967 0.00 9.500 19.478 19.372 N 3 3 3 3 3 3 Mean 1.48 3.75 46.130121.285 137.512 SD 0.619 0.00 20.580 53.331 53.418 N 3 3 3 3 3

In conclusion, single inhalation administration for 4 hours of PRINT-Treat a high average treprostinil dose of 1.409 mg/kg/day by Sprague-Dawleyrats was well tolerated as there were no significant test item relatedfindings. The exposure to treprostinil generally increased in a doseproportional manner between the low dose and the mid dose. The exposurebetween the mid and high dose increased in a slightly less than doseproportional manner. However, animals in the high dose group wereexposed to aerosol concentrations far below target for the last 16 to 26minutes of inhalation, which may account for the less than doseproportional increase in exposure. Based on these results, similar doselevels are recommended for the following definitive PK study. Bloodsampling time points during the test item inhalation period may beadjusted so as to better characterize exposure during test itemadministration.

Introduction

The objective of the study was to determine the pharmacokinetic profileof treprostinil in male Sprague Dawley rats when administered as thetest item, PRINT Treprostinil dry powder (PRINT-Tre), as a single 4 hourinhalation at targeted dose levels of 0.15, 0.75, and 1.5 mg/kg. Resultsfrom this study will be used to determine appropriate dose levels andsampling time points for a definitive PK bridging study.

The study was not performed in compliance with GLP regulations butfollowed appropriate Standard Operating Procedures (SOPs).

Experimental Design

The test item was administered to groups of rats by inhalationadministration for one day as described in the table below:

Targeted Total Inhaled Dose Level of Targeted Aerosol Targeted AerosolNo. of Group Group Treprostinil Concentration of Concentration ofAnimals No. Designation (mg/kg/day)^(a) Treprostinil (μg/L) Trehalose(μg/L) Males 1 Low Dose 0.15 1 130.7 3 2 Mid Dose 0.75 5 653.5 3 3 HighDose 1.5 10 1306.9 3 ^(a)= Targeted aerosol concentrations werecalculated based on an estimated body weight of 0.250 kg

Following dosing, a series of 6 blood samples for pharmacokineticevaluation were taken.

-   -   Characterization of Test Item

Test item*: Identity: PRINT Treprostinil Content: 92.75% of Trehalose,4% of Leucine, 2% of Tween80, 0.26% of NA Citrate Dihydrate, 0.25% ofNaCl: 0.74% of Treprostinil sodium (0.67% treprostinil) StorageConditions: Cool (2 to 8° C.), protect from moisture (e.g., dessicant)Handling Precautions: Standard laboratory precautions. Handle under dryconditions (Relative Humidity ≤23%) Supplier: Liquidia Technologies Inc.

Treatment

Acclimatization to Exposure System

Before the animals were exposed to the aerosol of the test item, ratswere accustomed to the restraint procedure over a period of 3 days. Theanimals were gradually accustomed to restraint in the dosing tubes usedduring the exposures up to the duration that was used for aerosoladministrations.

Animal Exposure

Exposure system used: Flow-past rodent inhalation exposure system

Exposure method: Inhalation by nose-only exposure

Test Item type: Dry-Powder formulation

Generation method: Piston feed/rotating brush generator

Duration of exposure: 240 minutes

The target aerosol concentrations and dose levels were as follows:

Targeted Dose Level Targeted Aerosol Targeted Aerosol Group Group ofTreprostinil Concentration of Concentration of No. Designation(mg/kg/day)^(a) Treprostinil (μg/L) Trehalose (μg/L) 1 Low Dose 0.15 1130.7 2 Mid Dose 0.75 5 653.5 3 High Dose 1.5 10 1306.9 ^(a)= Targetaerosol concentrations were calculated based on an estimated body weightof 0.250 kg.

Estimation of Achieved Dose Levels

The target dose levels were estimated using the following formula:

$D_{L} = \frac{E_{c} \times {RMV} \times T}{BW}$

-   D_(L)=Achieved dose levels (mg/kg/day)-   E_(c)=Actual concentration delivered to the animals (mg/L air)-   RMV=Respiratory Minute Volume (L/min) according to the method of    Bide, Armour and Yee J. App. Toxicol., Vol. 20, 2000:    RMV(L/min)=0.499× BW (kg)^(0.809)-   T=Time, duration of daily exposure (min.)-   BW=Mean body weight (kg) during exposure period.    This estimation of total inhaled dose assumed 100% deposition within    the respiratory tract.

Inhalation Exposure System

The powder aerosol was produced using a piston feed/rotating brushgenerator. The aerosol produced was diluted as necessary to achieve thetarget aerosol concentration and discharged through a 40-mm diametertube into a flow-past inhalation exposure system. The airflow ratethrough the exposure system was monitored and recorded manually duringeach aerosol generation period. Airflow to the exposure system wascontrolled by the absolute volume of air supplying the generationapparatus using variable area flowmeters. Control of the aerosol exhaustflow from the animal exposure system was achieved using an exhaustvalve, and the overall balance of airflows in the exposure system wasmonitored using pressure gauges. The system provided a minimum of 1.0L/min to each animal exposure port and was balanced to ensure a slightpositive pressure at the site of the proposed animal exposure. Thisensured that there was no dilution of the generated aerosol. An equaldelivery of aerosol to each proposed exposure position was achieved byemploying a distribution network that was identical for each individualexposure position attached to the system.

Inhalation System Monitoring

Determinations of aerosol concentration, particle size distribution,oxygen concentration, relative humidity and temperature were measured onsamples collected from a representative port of the exposure chamber.The sample flow rates were precisely controlled using variable area flowmeters that were calibrated before use using a primary airflowcalibrator. The absolute volume of each aerosol concentration sample wasmeasured with a wet type gas meter.

Oxygen Concentration

The oxygen concentration of the generated atmosphere was measured onceduring each aerosol exposure. Oxygen concentrations of the exposureatmospheres were maintained between 19-23%.

Relative Humidity/Temperature

The temperature and relative humidity of the generated atmosphere weremeasured once during each aerosol exposure. Temperatures of the exposureatmospheres were maintained between 19-24° C.

Determination of Aerosol Concentration

At least one aerosol concentration filter sample was collected on glassfiber filter and weighed on each day in order to measure the gravimetricconcentration of the test item in the generated aerosol. The filtersamples were transferred to the analytical chemistry laboratory forchemical determination of Treprostinil and Trehalose concentrationsusing an analytical method (Study No. 41609 and Study No. 41635).

Determination of the Particle Size Distribution and Mass MedianAerodynamic Diameter (MMAD)

The distribution of particle size in the generated aerosols was measuredonce during each exposure by collecting samples into a 7-Stage MercerCascade Impactor. The MMAD and the Geometric Standard Deviation (GSD)were calculated based on the results obtained from the impactor using alog-probit transformation.

In-Life Observations

Mortality

Mortality checks were performed at least once a day during all phases ofthe study.

Clinical Observations

Cage-side clinical signs (ill health, behavioral changes etc.) wererecorded at least once daily during all phases of the study, except ondetailed clinical examination days, where the cage-side clinical signswere replaced by a DCE.

A detailed clinical examination of each rat was performed on arrival aspart of the health status, as well as on Day 1, prior to dosing.

Animal whose health status was judged to warrant additional evaluationwas examined by a Clinical Veterinarian.

Body Weights

Body weights were recorded for all animals once at arrival as per healthstatus, once prior to group assignment and on Day 1 (prior to dosing).

Pharmacokinetics

A series of 6 blood samples (approximately 0.3 mL each) was collectedfrom each rat on Day 1 at −15, 5, 15, 30, 75 and 105 minutes aftertreatment. Thus a total blood volume of 1.8 mL was taken from each ratduring the course of the study. For this purpose, each rat(unanesthetized) was bled by jugular venipuncture and the samples werecollected into tubes containing the anticoagulant, K₂EDTA. Tubes wereplaced on wet ice pending processing.

Following collection, the samples were centrifuged (2500 rpm for 10minutes at approximately 4° C.) and the resulting plasma was recoveredand stored frozen (≤−60° C.) in labeled tubes.

Deviations to the pharmacokinetic time points were noted in the raw dataand were made available with the samples. The location of bloodwithdrawal was noted in the raw data.

Non-compartmental analysis of treprostinil concentrations in plasma wereperformed by using the Phoenix WinNonlin 6.3 software.

The following configuration was used for the analysis:

-   -   Sampling Method: Sparse    -   AUC Calculation Method: Linear Trapezoidal with Linear        Interpolation    -   Lambda Z (λ_(z)) Method: Best fit for λz, Log regression    -   Weighting (λ_(z) calculation): Uniform

Pharmacokinetic parameters (including abbreviation and description foreach parameter) are described in the following table:

Parameters Abbreviation Unit* Area under the plasma drugconcentration-time curve AUC_(0-Tlast) μg*hr/mL from the time of dosingto the last quantifiable concentration Area under the plasma drugconcentration-time curve AUC_(INF) μg*hr/mL from the time of dosingextrapolated to infinity Terminal elimination half-life T_(1/2) hr Themaximum plasma concentration C_(max) μg/mL Time to maximum plasmaconcentration T_(max) hr *Different units may be presented in the studyreport

Data Evaluation and Statistics

Numeric and non-numeric data obtained during the study were reportedonly as individual values.

Results

Aerosol Concentrations

Achieved gravimetric test atmosphere concentrations were as follows:

Group Targeted Aerosol Achieved Mean Aerosol Coefficient of % of No.Concentration (mg\L) Concentration (mg\L) Variation (%) Target 1 0.1560.165 17.2 105.9 2 0.781 0.728 14.0 93.2 3 1.563 1.439 43.5 92.0

Achieved analytical test atmosphere concentrations for treprostinil wereas follows:

Group Targeted Aerosol Achieved Mean Aerosol Coefficient of % of No.Concentration (μg\L) Concentration (μg\L) Variation (%) Target 1 1 1.0617.6 105.9 2 5 4.72 13.9 94.4 3 10 9.39 43.6 93.9

Achieved analytical test atmosphere concentrations for trehalose were asfollows:

Group Targeted Aerosol Achieved Mean Aerosol Coefficient of % of No.Concentration (μg\L) Concentration (μg\L) Variation (%) Target 1 130.7150.46 18.8 115.1 2 653.5 664.85 15.0 101.7 3 1306.9 1298.81* 44.1 99.4*Last 2 aerosol concentrations samples for trehalose were estimated witha 92.79% difference from gravimetric data as analytical results were BLQ

The overall achieved gravimetric and analytical aerosol concentrationsfor all groups were within 16% of the targeted concentrations. Thegenerated atmospheres were considered stable over the treatment periodas % CV were all below 20%, except for Group 3. The increased % CV forGroup 3 was caused by the stoppage of the Rotating Brush Generator (RBG)due to lack of test item remaining in the canister with 26 minutes leftin the generation (16 minutes of dosing left for animal 3001A, 21minutes left for animal 3002A and 26 minutes left for animal 3003A).Though a new test item canister was installed on the RBG apparatus, theaerosol concentrations were much lower than targeted for the last 26minutes. However, the overall aerosol concentrations were stillconsidered acceptable for the study as there was a significantdifference in aerosol concentration between groups.

Dose Levels

Overall achieved doses for treprostinil are presented below:

Targeted Duration of Body Estimated % from Group Dose Levels ExposureWeight Achieved Doses Targeted Dose No. (mg/kg/day) (min) Animal (kg)(mg/kg/day) Level 1 0.15 240 1001 A 0.326 0.157 104.7 1002 A 0.309 0.159106.0 1003 A 0.314 0.158 105.3 Average 0.158 105.3 2 0.75 240 2001 A0.319 0.703 93.7 2002 A 0.308 0.708 94.4 2003 A 0.304 0.709 94.5 Average0.707 94.3 3 1.5 240 3001 A 0.322 1.396 93.1 3002 A 0.321 1.397 93.13003 A 0.281 1.433 95.5 Average 1.409 93.9

Average achieved dose levels for all groups were within 7% of thetargeted dose levels therefore the dose levels were consideredacceptable for the study as a clear dose differentiation between groupsfor each sex was achieved.

Particle Size Distribution

The average gravimetric particle size distribution measurement data wereas follows:

Cumulative % Less Than Stated Mean % Group Effective Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 1 95.8 92.4 82.4 45.4 32.4 21.5 12.3 0.0 1.2 2.28 93 2 86.8 83.3 76.039.4 28.0 15.4  8.9 0.0 1.5 2.56 85 3 94.0 90.9 77.3 30.3 13.4  8.9  5.30.0 1.6 2.06 90 MMAD = Mass median aerodynamic diameter GSD = Geometricstandard deviation.

The average chemical determination of particle size distribution fortreprostinil were as follows:

Cumulative % Less Than Stated Mean % Group Effective Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 1 96.4 93.9 83.3 42.8 28.1 16.5 6.9 0.0 1.3 2.06 94 2 91.7 88.5 81.137.9 25.8 11.8 4.9 0.0 1.5 2.15 90 3 95.2 91.5 76.6 25.7  8.8  5.1 1.80.0 1.7 1.86 91 MMAD = Mass median aerodynamic diameter GSD = Geometricstandard deviation.

The average chemical determination of particle size distribution fortrehalose were as follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 1 95.8 91.6 81.5 38.4 24.1 13.0 4.2 0.0 1.4 2.01 93 2 94.4 88.9 83.331.5 26.0 11.1 5.6 0.0 1.4 2.11 91 3 95.2 90.4 77.0 25.5 9.6 4.8 0.0 0.01.8 1.65 94 MMAD = Mass median aerodynamic diameter GSD = Geometricstandard deviation.

The particle size distribution was considered respirable for this studyas the MMADs were below 4 μm and the GSD were within 1.5 and 3.

Exposure Chamber Conditions

Exposure chamber conditions from the reported aerosol concentrationexposures are summarized below.

Group Humidity Temperature Oxygen Concentration No. (% RH) (° C.) (%) 131.4 21.2 20.9 2 34.2 21.7 20.9 3 26.2 20.7 20.9

Exposure atmosphere oxygen concentrations, temperature and relativehumidity ranges were considered acceptable on all occasions.

Mortality

There were no mortalities during the study.

Clinical Signs

There were no adverse clinical signs observed during the study.

Slight decreased activity, piloerection and partially closed eyes wereseen in animal 3001A right before the 15 minute time point. Howeverthese were not observed afterwards and were not observed in any otheranimal therefore were not deemed test item related.

Body Weight

Body weights were performed for dose level calculation purposes.

Pharmacokinetics

Following the administration of PRINT-Tre at all achieved dose levels,mean C_(max) ranged from 6.800 to 46.133 ng/mL. The mean maximum plasmaconcentration (T_(max)) was reached at 3.75 hour (15 minutes before endof dosing) for all groups. The mean AUC_(0-Tlast) (AUC_(INF)) rangedfrom 17.320 (18.335) to 121.258 (137.512) hr*ng/mL. Following T_(max),the treprostinil plasma concentrations declined gradually with anestimated mean T_(1/2) ranging from 1.01 to 1.68 hours.

Over the dose range, exposure to treprostinil (based on C_(max),AUC_(0-Tlast) and AUC_(INF)) generally increased in dose proportionalmanner between the low dose (0.158 mg/kg) and the mid dose (0.707mg/kg). When dose level increased 4.5-fold from low to mid dose, C_(max)and AUC_(0-Tlast) increased 4.7-fold. Treprostinil exposure between themid dose (0.707 mg/kg) and high dose (1.409 mg/kg) increased in aslightly less than dose proportional manner (2-fold increase in dosewith a 1.4−(C_(max)) to 1.5-fold (AUC_(0-Tlast)) increase in exposure).However, because animals in the high dose group were exposed to aerosolconcentrations far below target for the last 16 to 26 minutes of theexposure period, exposure levels may have been effected and couldaccount for the less than dose proportional increase in exposure.

Conclusion

Single inhalation administration for 4 hours of PRINT-Tre at a highaverage treprostinil dose of 1.409 mg/kg/day to Sprague-Dawley rats waswell tolerated as there were no significant test item related findings.The exposure to treprostinil generally increased in a dose proportionalmanner between the low dose and the mid dose. The exposure between themid and high dose increased in a slightly less than dose proportionalmanner. However, animals in the high dose group were exposed to aerosolconcentrations far below target for the last 16 to 26 minutes ofinhalation, which may account for the less than dose proportionalincrease in exposure. Based on these results, similar dose levels arerecommended for the following definitive PK study. Blood sampling timepoints during the test item inhalation period may be adjusted so as tobetter characterize exposure during test item administration.

Non-GLP, Single-Dose, Inhalation, Comparative PK Study of LIQ861 andNebulized Treprostinil in Rats (Study 75658)

This study evaluated and compared the PK profile of LIQ861 totreprostinil (nebulized) to establish a bridge between the twoformulations.

The non-GLP, single administration by inhalation, PK study oftreprostinil in rats (Study 75658) has been completed by Liquidia(referred to as the definitive PK bridging study). This study comparedthe systemic exposure of LIQ861 versus nebulized liquid treprostinilsodium. The observed systemic exposures revealed no meaningfuldifferences between formulations, providing a bridge between the LIQ861formulation and the marketed Tyvaso formulation and thereby permittinguse of Tyvaso nonclinical toxicology studies to support the LIQ861formulation per the 505(b)(2) pathway.

In Study 75658, systemic exposure of LIQ861 versus nebulizedtreprostinil sodium was compared in rats. LIQ861 was delivered over a4-hour exposure period at total delivered dose levels of 0.273, 0.762,and 1.50 mg/kg body weight. Nebulized treprostinil sodium was deliveredat a single dose level (0.785 mg/kg total delivered dose) for the sameexposure period (4 hours) as LIQ861. Blood was collected for plasmaanalysis of treprostinil concentrations at 30 and 60 minutes followingthe start of administration, immediately post-administration (240 min),and at 5, 15, 30, 75, and 105 minutes following the end ofadministration.

Pharmacokinetic parameters from Study 75658. Individual plasmaconcentrations of treprostinil ranged from 0.345 to 67.4 ng/mL. Maximumplasma concentration was reached 0.5 to 4 hours after the start of the4-hour exposure period. Maximum concentration (Cmax) and area under thecurve (AUC) values were similar between males and females withintreatment groups. Dose-related increases in Cmax and AUC values wereobserved for the three LIQ861 dose groups. Relative bioavailability ofLIQ861 compared to nebulized treprostinil based on dose normalizedAUC-time curve extrapolated to time infinity (AUCinf) ranged from 1.2 to2.2.

Summary of Mean Noncompartmental PK Parameters by Treatment and Sex forStudy 75658

Achieved Mean Dose Type of Level t_(1/2) T_(max) C_(max) AUC_(0-Tlast)AUC_(INF) inhalation Group (mg/kg) Sex R² (hr) (hr) (ng/mL) (hr*ng/mL)(hr*ng/mL) Treprostinil 1 0.785 Female 0.99 0.59 4.00 16.2 62.4 63.5Sodium Male 0.97 0.77 0.50 16.5 59.3 60.6 (Nebulized) Dry Powder 2 0.273Female 0.92 1.77 4.00 5.38 22.2 24.2 (PRINT Male 1.00 0.73 1.00 7.1826.9 27.4 treprostinil) 3 0.762 Female 0.97 0.66 4.00 32.8 107 110 Male0.90 0.95 4.00 54.4 144 149 4 1.498 Female 0.84 0.67 0.50 44.5 174 182Male 1.00 0.90 4.00 44.1 143 148 Abbreviations: C_(max), maximalconcentration; T_(max), time of maximal concentration; AUC_(last), areaunder the concentration-time curve to the last measured timepoint;t_(1/2), half-life; AUC_(inf), area under the concentration-time curveextrapolated to time infinity. PRINT treprostinil = LIQ861DP-intermediate.Calculated Relative Bioavailability (Combined Genders) Based on AUCinf(Dose Corrected) from Study 75658

Dose Mean Frel Level AUCinf (PRINT/ Group Treatment (mg/kg) (hr*ng/mL)Treprostinil) 1 Treprostinil 0.785 62.1 NA 2 PRINT- 0.273 25.8 1.20treprostinil Low 3 PRINT-treprostinil Mid 0.762 129 2.15 4 PRINT- 1.498165 1.39 treprostinil High Abbreviations: AUCinf, area under theconcentration-time curve extrapolated to time infinity; Frel, relativebioavailability; NA, not applicable. PRINT treprostinil = LIQ861DP-intermediate.

Summary: Study 75658

The objectives of the study were to determine the pharmacokinetic (PK)profile of Treprostinil in Sprague-Dawley rats when administered asPRINT-Treprostinil (PRINT-Tre) by 4-hour inhalation at 0.15, 0.75, and1.5 mg/kg, to determine the PK profile of Treprostinil in Sprague-Dawleyrats when administered as nebulized Treprostinil sodium in solution (Tresolution) by 4-hour inhalation at 0.75 mg/kg and to compare the PKprofiles of Treprostinil when administered as PRINT-Tre and Tresolution.

The test item was administered once to 6 male and 6 female rats pergroup by nose-only inhalation for 4 hours as described in the tablebelow:

Achieved Mean Total Inhaled Dose Level of Achieved Aerosol AchievedAerosol Group Treprostinil Concentration of Concentration of No. GroupDesignation (mg/kg/day) Treprostinil (μg/L) Trehalose (μg/L) 1 TreSolution 0.785 5.07 0 2 PRINT-Tre (Low Dose) 0.273 1.76 254.84 3PRINT-Tre (Mid Dose) 0.762 4.95 719.53 4 PRINT-Tre (High Dose) 1.4989.73 1394.33

Assessments of mortality, clinical signs and body weights wereperformed. Pharmacokinetic samples were collected and the analysis ofthese samples was performed.

No mortality occurred and no clinical signs were observed.

The overall achieved aerosol concentrations for all groups were within10% of the targeted concentrations gravimetrically and for bothtreprostinil and trehalose, except for Group 2 which were significantlyabove the targeted concentrations (76 to 95%). Corresponding averageachieved dose levels for all groups were within 5% of the targeted doselevels, except for Group 2 which was 82% above the targeted dose level.However, the dose levels were considered acceptable for the study as aclear dose differentiation between groups for each sex was achieved.

The particle size MMADs from Groups 2 to 4 were between 1.7 and 2.0 μmgravimetrically (GSD 1.90 to 2.67); for both treprostinil and trehalose,chemical particle size distribution ranged from 1.6 to 1.8 μm with thecorresponding GSDs between 1.89 and 2.24. The particle size MMAD forGroup 1 was 0.5 μm with a corresponding GSD of 2.60. The particle sizedistribution was considered respirable.

With administration of PRINT-Tre at an achieved dose level of 0.273mg/kg, 0.762 mg/kg or 1.498 mg/kg, plasma exposure to treprostinil wasgenerally similar in both sexes; however, exposure was slightly lower infemales than males at the mid-dose level and slightly higher in femalesthan males at the high-dose level.

Based on AUC_(0-Tlast), AUC_(INF) and C_(max), values for both sexes,plasma exposure increased more than proportionally between the low- andmid-dose levels. But between the mid- and high-dose levels, plasmaexposure increased less than proportionally for females and there was noincrease in the exposure for males. The maximum mean treprostinil plasmaconcentration (T_(max)) was at the end of inhalation for both sexes,except for low-dose males and high-dose females, where mean T_(max) wasat 1 and 0.5 hours after inhalation began, respectively.

At the low-dose level, mean treprostinil plasma concentration wassimilar after 0.5, 1, or 4 hours of inhalation exposure to PRINT-Tre,suggesting that steady state was achieved within the first 30 minutes ofexposure. The same was true for females at the high-dose level; however,for males at the high-dose level and for both sexes at the mid-doselevel, mean treprostinil plasma concentration was greater at the end ofinhalation than after one hour of inhalation. When inhalation ended,treprostinil plasma concentrations declined gradually. Given the degreeof individual variation, the estimated mean T_(1/2) values were similarat all dose levels and ranged from 0.7 to 1.8 hours in males and 0.7 to1.0 hours in females.

For Tre solution, with an administration at 0.785 mg/kg, plasma exposureto treprostinil was generally similar in both sexes. The maximum meantreprostinil plasma concentration (T_(max)) was at the end ofinhalation. Mean treprostinil plasma concentration was similar after0.5, 1, or 4 hours of inhalation exposure to Tre solution, suggestingthat steady state was achieved within the first 30 minutes of exposure.When inhalation ended, treprostinil plasma concentrations declinedgradually, with estimated mean T_(1/2) values of 0.6 hours in males and0.8 hours in females.

Administration of PRINT-Tre and Tre solution at nearly equivalent doselevels (0.76 and 0.79 mg/kg, respectively) resulted in plasma exposuresto treprostinil that were greater with PRINT-Tre than with Tre solution.Specifically, mean AUC_(0-Tlast) was approximately twice as high (126versus 61 h*ng/mL, respectively) and mean C_(max) was three times ashigh (44 versus 16 ng/mL, respectively). As would be expected, oncetreprostinil entered the systemic circulation, it was cleared fromplasma at a similar rate, with mean T_(1/2) values of 0.7 to 1.0 hoursfor PRINT-Tre and 0.6 to 0.8 hours for Tre solution.

In conclusion, single inhalation administration for 4 hours of PRINTTreprostinil at a high average dose of 1.498 mg/kg/day to Sprague-Dawleyrats was well tolerated as there were no test item related findings. Atan equivalent dose level, plasma exposures to treprostinil was greaterwith PRINT-Tre than with Tre solution; specifically, mean AUC_(0-Tlast)was approximately twice as great and mean C_(max) was three times asgreat. As would be expected, once treprostinil entered the systemiccirculation, it was cleared from plasma at a similar rate regardless ofhow it was administered.

The objectives of the study were to:

-   -   1. Determine the pharmacokinetic (PK) profile of Treprostinil in        Sprague-Dawley rats when administered as PRINT-Treprostinil        (PRINT-Tre) by 4-hour inhalation at 0.15, 0.75, and 1.5 mg/kg.    -   2. Determine the PK profile of Treprostinil in Sprague-Dawley        rats when administered as nebulized Treprostinil sodium in        solution (Tre solution) by 4-hour inhalation at 0.75 mg/kg.    -   3. Compare the PK profiles of Treprostinil when administered as        PRINT-Tre and Tre solution.

Experimental Design

The test items were administered to groups of rats by a 4-hourinhalation administration as described in the table below:

Targeted Total Targeted Inhaled Dose Aerosol Level of Concentration ofTargeted Aerosol Group Group Treprostinil Treprostinil Concentration ofNo. of Animals No. Designation (mg/kg/day) (μg/L) ^(a) Trehalose (μg/L)^(a) Males Females 1 Tre Solution 0.75 5 0 6 6 2 PRINT-Tre 0.15 1 130.76 6 3 PRINT-Tre 0.75 5 653.5 6 6 4 PRINT-Tre 1.5 10 1306.9 6 6 ^(a) =Target aerosol concentrations were calculated based on an estimated bodyweight of 0.250 kg

During and after the inhalation period, a series of 8 blood samples forpharmacokinetic evaluation were taken.

Justification for Selection of Route of Administration, Species and DoseLevels

The route of administration was chosen because it is the intended humantherapeutic route.

The rat was selected because it is a rodent species recommended byvarious regulatory authorities. Background data are available. Also,rats were used as the test system for previous toxicity studies withTreprostinil sodium solution that supported development and approval ofthat product. Using rats in the current study allowed comparison withthe previous studies.

The high-dose level for PRINT-Tre was the feasible dose attainable basedon technical aerosol trials with the test item (Study No. 41610).

The low- and mid-dose levels for PRINT-Tre were selected on the basis ofa previous pilot PK study in rats (Study No. 75670).

The dose level for Tre solution was selected to match the mid-dose levelof PRINT-Tre to allow direct comparison.

Characterization of Test Items

Content: 92.79% of Trehalose, 4% of Leucine, 2% of Tween80, 0.26% of NACitrate, 0.24% of NaCl:0.71% of Treprostinil Storage Cool (2 to 8° C.),protected from Conditions: moisture (e.g., dessicant) Handling Standardlaboratory precautions. Precautions: Handle under dry conditions(Relative Humidity ≤ 23%) Supplier: Liquidia Technologies Inc. Test item2*: Identity: Treprostinil Sodium Description: White or pale yellowishpowder Batch No.: TN115E010 Expiry Date: May 28, 2017 Purity: 101.49%Storage Cool (2 to 8° C.) Conditions: Handling Standard laboratoryprecautions Precautions: Supplier: Yonsung Fine Chemicals Co., LTD

Preparation of Test Item

PRINT-Tre was used as provided by the Sponsor. A glove box undernitrogen was used for handling, aliquoting or packing of the canisters.Relative humidity (RH) inside the glove box was monitored and recordedusing a hygrometer and was kept below 23% RH.

For Group 1, the treprostinil sodium was dissolved in purified water toachieve the desired formulation concentration. A representative sample(0.5 mL in duplicate) was collected to verify the formulationconcentration of Treprostinil in the formulation.

Treatment

Acclimatization to Exposure System

Before the animals were exposed to the aerosol of the test item, ratswere accustomed to the restraint procedure over a period of 3 days. Theanimals were gradually accustomed to restraint in the dosing tubes usedduring the exposures up to the duration that was used for aerosoladministrations.

Animal Exposure

Exposure system used: Flow-past rodent inhalation exposure system

Exposure method: Inhalation by nose-only exposure

Test Item type: Solution (Group 1), Dry Powder (Groups 2 to 4)

Generation method: Nebulization (Group 1) and Piston feed/rotating brushgenerator (Group 2 to 4)

Duration of exposure: 240 minutes

The target aerosol concentrations and dose levels were as follows:

Targeted Dose Level Targeted Aerosol Targeted Aerosol Group ofTreprostinil Concentration of Concentration of No. Group Designation(mg/kg/day) Treprostinil (μg/L) ^(a) Trehalose (μg/L) 1 Tre solution0.75 5 0 2 PRINT-Tre (Low Dose) 0.15 1 130.7 3 PRINT-Tre (Mid Dose) 0.755 653.5 4 PRINT-Tre (High Dose) 1.5 10 1306.9 ^(a) = Target aerosolconcentrations were calculated based on an estimated body weight of0.250 kg.

Estimation of Achieved Dose Levels

The target dose levels were estimated using the following formula:

$D_{L} = \frac{E_{c} \times {RMV} \times T}{BW}$

-   D_(L)=Achieved dose levels (mg/kg/day)-   E_(c)=Actual concentration delivered to the animals (mg/L air)-   RMV=Respiratory Minute Volume (L/min) according to the method of    Bide, Armour and Yee 2000 J. App. Toxicol., Vol. 20: RMV    (L/min)=0.499×BW (kg)° 0.809-   T=Time, duration of daily exposure (min.)-   BW=Mean body weight (kg) during exposure period.    This estimation of total inhaled dose assumed 100% deposition within    the respiratory tract.

Inhalation Exposure System

The powder aerosol for Groups 2 to 4 was produced using a pistonfeed/rotating brush generator while the liquid aerosol for Group 1 wasproduced by metering the flow of the formulation to a clinical nebulizer(Sidestream). The aerosol produced was diluted as necessary to achievethe target aerosol concentration and discharged through a 40-mm diametertube into a flow-past inhalation exposure system. The airflow ratethrough the exposure system was monitored and recorded manually duringeach aerosol generation period. Airflow to the exposure system wascontrolled by the absolute volume of air supplying the generationapparatus using variable area flowmeters. Control of the aerosol exhaustflow from the animal exposure system was achieved using an exhaustvalve, and the overall balance of airflows in the exposure system wasmonitored using pressure gauges. The system provided a minimum of 1.0L/min to each animal exposure port and was balanced to ensure a slightpositive pressure at the site of the animal exposure. This ensured thatthere was no dilution of the generated aerosol. An equal delivery ofaerosol to each exposure position was achieved by employing adistribution network that was identical for each individual exposureposition attached to the system.

Inhalation System Monitoring

Determinations of aerosol concentration, particle size distribution,oxygen concentration, relative humidity and temperature were measured onsamples collected from a representative port of the exposure chamber.The sample flow rates were precisely controlled using variable area flowmeters that were calibrated before use using a primary airflowcalibrator. The absolute volume of each aerosol concentration sample wasmeasured with a wet type gas meter.

Oxygen Concentration

The oxygen concentration of the generated atmosphere was measured onceduring each aerosol exposure. Oxygen concentrations of the exposureatmospheres were maintained between 19-23%.

Relative Humidity/Temperature

The temperature and relative humidity of the generated atmosphere weremeasured once during each aerosol exposure. Temperatures of the exposureatmospheres were maintained between 19-24° C.

Determination of Aerosol Concentration

At least one aerosol concentration filter sample was collected for allgroups on each aerosol generation. The filter samples from Groups 2 to 4were weighed in order to measure the gravimetric concentration of thetest item in the generated aerosol. The filter samples were transferredto the analytical chemistry laboratory for chemical determination ofTreprostinil and Trehalose concentrations. The filter samples for Group1 were not weighed gravimetrically and were only transferred to theanalytical laboratory for determination of Treprostinil concentrations.The analysis in the analytical laboratory was performed using ananalytical method (Study No. 41609).

Determination of the Particle Size Distribution and Mass MedianAerodynamic Diameter (MMAD)

The distribution of particle size in the generated aerosols was measuredonce for Groups 1 to 4 by collecting samples into a 7-Stage MercerCascade Impactor. All sample substrates obtained from Groups 2 to 4 wereweighed gravimetrically and then transferred to the analytical chemistrylaboratory for chemical determination of particle size of aerosolizedTreprostinil and Trehalose. All sample substrates obtained from Group 1were only transferred to the analytical laboratory for chemicaldetermination of particle size of aerosolized Treprostinil. The analysisin the analytical laboratory was performed using an analytical method(Study No. 41609).

The MMAD and the Geometric Standard Deviation (GSD) were calculatedbased on the results obtained from the impactor using a log-probittransformation.

Reporting of Analytical Results

The analytical report containing the results from the filter andparticle size distribution sample analyses were prepared. Any samplesnot employed in the primary analysis or any remaining sample from theprimary analysis were retained until it was determined by the analystand Study Director that it was not be required for confirmatoryanalysis. These samples were discarded and their disposition recorded inthe raw data.

In-Life Observations

Mortality

Mortality checks were performed at least once a day during all phases ofthe study.

Clinical Observations

Cage-side clinical signs (ill health, behavioral changes etc.) wererecorded at least once daily during all phases of the study, except ondetailed clinical examination days, where the cage-side clinical signswere replaced by a DCE.

A detailed clinical examination of each rat was performed on arrival aspart of the health status, as well as on Day 1, prior to dosing.

Body Weights

Body weights were recorded for all animals once at arrival as per healthstatus, once prior to group assignment and on Day 1 (prior to dosing).

Pharmacokinetics

A series of 8 blood samples (approximately 0.3 mL each) was collected 30minutes and 1 hour after exposure began, immediately after exposureended (IPE), and again at 5, 15, 30, 75 and 105 minutes post-dosing asper the table below. Thus a total blood volume of 1.2 mL was taken fromeach rat during the course of the study. For this purpose, each rat(unanesthetized) was bled by jugular venipuncture and the samples werecollected into tubes containing the anticoagulant, K₂EDTA. Tubes wereplaced on wet ice pending processing.

Toxicokinetic time point 1 hour Group Number of 30 min post 5 min 15 min30 min 75 min 105 min Number animals/sex post start start IPE post endpost end post end post end post end 1 3+ ✓ ✓ ✓ ✓ 3 # ✓ ✓ ✓ ✓ 2 3+ ✓ ✓ ✓✓ 3 # ✓ ✓ ✓ ✓ 3 3+ ✓ ✓ ✓ ✓ 3 # ✓ ✓ ✓ ✓ 4 3+ ✓ ✓ ✓ ✓ 3 # ✓ ✓ ✓ ✓ +animals with the lowest identification numbers # animals with thehighest identification numbers

Following collection, the samples were centrifuged (2500 rpm for 10minutes at approximately 4° C.) and the resulting plasma was recoveredand stored frozen (≤−60° C.) in labeled tubes.

Deviations to the pharmacokinetic time points were noted in the raw dataand were made available with the samples. The location of bloodwithdrawal was noted in the raw data.

The plasma analysis was performed and the bioanalytical data wasprepared for inclusion in the final report.

The pharmacokinetic parameters were calculated and the non-compartmentalanalysis of PRINT-Tre and Tre solution treprostinil concentrations inplasma was performed by using the Phoenix WinNonlin 6.3 software.

The following configuration was used for the analysis:

-   -   Sampling Method: Sparse    -   AUC Calculation Method: Linear Trapezoidal with Linear        Interpolation    -   Lambda Z (λ_(z)) Method: Best fit for λz, Log regression    -   Weighting (λ_(z) calculation): Uniform

Pharmacokinetic parameters (including abbreviation and description foreach parameter) were described in the following table:

Parameters Abbreviation Unit* Area under the plasma drug concentration-AUC_(0-Tlast) μg*hr/mL time curve from the time of dosing to the lastquantifiable concentration Area under the plasma drug concentration-AUC_(INF) μg*hr/mL time curve from the time of dosing extrapolated toinfinity Terminal elimination half-life T_(1/2) hr The maximum plasmaconcentration C_(max) μg/mL Time to maximum plasma concentration T_(max)hr

Data Evaluation and Statistics

Numeric and non-numeric data obtained during the study were reportedonly as individual values.

Results

Formulation Analysis

Formulation concentration for Group 1 was as follows:

Average Targeted Average Measured Concentration Concentration % ofTargeted Group No. (mg/mL) (mg/mL) Concentration 1 0.50 0.492 98.4

The formulation concentration for Group 1 was within 2% of the targetedconcentration therefore the formulation concentration was consideredacceptable for the study.

Aerosol Concentrations

Achieved gravimetric test atmosphere concentrations were as follows:

Targeted Aerosol Achieved Mean Aerosol Coefficient of GroupConcentration Concentration Variation % of No. (mg\L) (mg\L) (%) Target2 0.156 0.283 55.5 181.4 3 0.781 0.814 16.3 104.2 4 1.563 1.548 21.999.0

Achieved chemical test atmosphere concentrations for treprostinil wereas follows:

Targeted Aerosol Achieved Mean Aerosol Coefficient of GroupConcentration Concentration Variation % of No. (μg\L) (μg\L) (%) Target1 5 5.07 3.6 101.4 2 1 1.76 55.2 176.3 3 5 4.95 19.8 99.1 4 10 9.73 22.397.3

Achieved chemical test atmosphere concentrations for trehalose were asfollows:

Targeted Achieved Coefficient Aerosol Mean Aerosol of GroupConcentration Concentration Variation % of No. (μg\L) (μg\L) (%) Target2 130.7 254.84 54.4 195.0 3 653.5 719.53 20.5 110.1 4 1306.9 1394.3323.2 106.7

The overall achieved aerosol concentrations for all groups were within10% of the targeted concentrations gravimetrically and for bothtreprostinil and trehalose, except for Group 2 which were significantlyabove the targeted concentrations (76% and 95% for treprostinil andtrehalose, respectively). The generated atmospheres were consideredstable over the treatment period except for Group 2 (CV %˜54%). However,the overall aerosol concentrations were still considered acceptable forthe study as there was a significant difference in aerosol concentrationbetween groups.

Achieved Dose Levels

Overall achieved doses for treprostinil are presented below:

Targeted Duration of Body Estimated % from Group Dose Level ExposureWeight Achieved Doses Targeted Dose No. (mg/kg/day) (min) Sex (kg)(mg/kg/day) Level 1 0.75 240 Male 0.308 0.760 101.4 Female 0.212 0.817108.9 Combined 0.260 0.785 104.7 2 0.15 240 Male 0.301 0.265 176.7Female 0.211 0.284 189.1 Combined 0.256 0.273 182.3 3 0.75 240 Male0.321 0.737 98.2 Female 0.215 0.795 106.0 Combined 0.268 0.762 101.6 41.5 240 Male 0.317 1.451 96.7 Female 0.219 1.557 103.8 Combined 0.2681.498 99.9

Average achieved dose levels for all groups were within 5% of thetargeted dose levels, except for Group 2 which was 82% above thetargeted dose level. However, the dose levels were considered acceptablefor the study as a clear dose differentiation between groups for eachsex was achieved.

Particle Size Distribution

The average gravimetric particle size distribution measurement data wereas follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 2 87.9 80.0 61.0 31.2 21.8 16.2 9.1 0.0 1.7 2.67 80 3 90.7 85.0 66.924.9 14.8 9.0 3.0 0.0 1.8 2.12 85 4 91.7 84.0 61.6 23.5 8.1 4.3 0.8 0.02.0 1.90 86 MMAD = Mass median aerodynamic diameter GSD = Geometricstandard deviation.

The average chemical determination of particle size distribution fortreprostinil were as follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 1 95.9 95.7 95.4 95.1 85.1 50.1 22.0 0.0 0.5 2.60 98 2 94.0 86.3 64.429.3 19.8 14.1 6.1 0.0 1.6 2.24 87 3 95.3 90.3 71.5 25.9 15.2 9.3 2.70.0 1.6 1.97 90 4 94.1 88.4 64.3 23.9 8.2 4.4 1.5 0.0 1.8 1.89 88 MMAD =Mass median aerodynamic diameter GSD = Geometric standard deviation.

The average chemical determination of particle size distribution fortrehalose were as follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 2 94.3 86.7 63.8 26.4 16.8 14.1 6.1 0.0 1.6 2.22 87 3 96.0 92.0 72.322.2 12.0 8.0 4.0 0.0 1.6 1.97 90 4 95.7 91.4 68.0 27.9 12.8 8.6 4.3 0.01.6 2.00 90 MMAD = Mass median aerodynamic diameter GSD = Geometricstandard deviation.

The particle size distribution was considered respirable for this studyas the MMADs were below 4 μm and the GSD were within 1.5 and 3.

Exposure Chamber Conditions

Exposure chamber conditions from the reported aerosol concentrationexposures are summarized below.

Temperature Oxygen Concentration Group No. Humidity (% RH) (° C.) (%) 158.5 21.0 20.9 2 35.1 21.6 20.9 3 39.0 21.5 20.9 4 39.4 21.2 20.9

Exposure atmosphere oxygen concentrations, temperature and relativehumidity ranges were considered acceptable on all occasions.

Mortality

There were no mortalities during the study.

Clinical Signs

There were no clinical signs observed during the study.

Body Weight

Body weights were performed for dose level calculation purposes.

Pharmacokinetics

With administration of PRINT-Tre at an achieved dose level of 0.273mg/kg, 0.762 mg/kg or 1.498 mg/kg, plasma exposure to treprostinil wasgenerally similar in both sexes; however, exposure was slightly lower infemales than males at the mid-dose level and slightly higher in femalesthan males at the high-dose level.

Based on AUC_(0-Tlast), AUC_(INF) and C_(max), values for both sexes,plasma exposure increased more than proportionally between the low- andmid-dose levels. But between the mid- and high-dose levels, plasmaexposure increased less than proportionally for females and there was noincrease in the exposure for males. The maximum mean treprostinil plasmaconcentration (T_(max)) was at the end of inhalation for both sexes,except for low-dose males and high-dose females, where mean T_(max) wasat 1 and 0.5 hours after inhalation began, respectively.

At the low-dose level, mean treprostinil plasma concentration wassimilar after 0.5, 1, or 4 hours of inhalation exposure to PRINT-Tre,suggesting that steady state was achieved within the first 30 minutes ofexposure. The same was true for females at the high-dose level; however,for males at the high-dose level and for both sexes at the mid-doselevel, mean treprostinil plasma concentration was greater at the end ofinhalation than after one hour of inhalation. These data are summarizedbelow.

Males Females Dose (mg/kg) = 0.273 0.762 1.498 0.273 0.762 1.498 0.5hours of inhalation 6.1 22 26 4.2 18 44 1 hour of inhalation 7.2 21 275.1 19 35 4 hours of inhalation 5.4 54* 44{circumflex over ( )} 5.4 33**44 *Individual values were 33, 63, and 67 ng/mL {circumflex over( )}Individual values were 34, 48, and 50 ng/mL **Individual values were22, 27, and 49 ng/mL

When inhalation ended, treprostinil plasma concentrations declinedgradually. Given the degree of individual variation, the estimated meanT_(1/2) values were similar at all dose levels and ranged from 0.7 to1.8 hours in males and 0.7 to 1.0 hours in females.

For Tre solution, with an administration at 0.785 mg/kg, plasma exposureto treprostinil was generally similar in both sexes.

The maximum mean treprostinil plasma concentration (T_(max)) was at theend of inhalation. Mean treprostinil plasma concentration was similarafter 0.5, 1, or 4 hours of inhalation exposure to Tre solution,suggesting that steady state was achieved within the first 30 minutes ofexposure. These data are summarized below.

Males Females 0.5 hours of inhalation 17 12   1 hour of inhalation 11 14  4 hours of inhalation 16 16

When inhalation ended, treprostinil plasma concentrations declinedgradually, with estimated mean T_(1/2) values of 0.6 hours in males and0.8 hours in females.

Administration of PRINT-Tre and Tre solution at nearly equivalent doselevels (0.76 and 0.79 mg/kg, respectively) resulted in plasma exposuresto treprostinil that were greater with PRINT-Tre than with Tre solution.Specifically, mean AUC_(0-Tlast) was approximately twice as high (126versus 61 h*ng/mL, respectively) and mean C_(max) was three times ashigh (44 versus 16 ng/mL, respectively). As would be expected, oncetreprostinil entered the systemic circulation, it was cleared fromplasma at a similar rate, with mean T_(1/2) values of 0.7 to 1.0 hoursfor PRINT-Tre and 0.6 to 0.8 hours for Tre solution.

Conclusion

Single inhalation administration for 4 hours of PRINT Treprostinil at ahigh average dose of 1.498 mg/kg/day to Sprague-Dawley rats was welltolerated as there were no test item related findings. At an equivalentdose level, plasma exposures to treprostinil was greater with PRINT-Trethan with Tre solution; specifically, mean AUC_(0-Tlast) wasapproximately twice as great and mean C_(max) was three times as great.As would be expected, once treprostinil entered the systemiccirculation, it was cleared from plasma at a similar rate regardless ofhow it was administered.

Non-GLP, 7-Day, Repeat-Dose, Range-Finding (DRF), Inhalation Study withLIQ861 in Rats (Study 75654)

Results from the completed comparative PK study will be used to selectdose levels to be tested in this DRF study, which will evaluate localtoxicity in the respiratory tract as well as systemic treprostiniltoxicity. Results will be used to select appropriate dose levels for a2-week GLP repeat-dose toxicology study in rats.

Summary: Study 75654

The objectives of the study were to evaluate the toxicity of the testitem, PRINT Treprostinil, and the excipients that make up the controlitem, PRINT Placebo, when administered to Sprague-Dawley rats bynose-only inhalation for 4 hours a day for 7 days. Results were used tohelp select dose levels for a subsequent 14-day GLP inhalationtoxicology study.

Groups of 6 rats (3/sex) were exposed by 4-hour inhalation daily for 7days to air, PRINT Placebo, or PRINT Treprostinil at treprostinil doselevels of approximately 170, 680, or 1370 μg/kg, as described in thetable below:

Mean Dose Levels and Concentrations^(a) Treprostinil Trehalose LeucineGroup Group Dose Level Aerosol Conc. Dose Level Aerosol Conc. Dose LevelNo. Designation (ug/kg/day) (μg/L) (mg/kg/day) (μg/L) (mg/kg/day)^(b) 1Air Control 0 0 0 0 0 2 Placebo 0 0 281.2 1832.13 12.0 Control^(b) 3PRINT-Tre 170 1.10 33.1 216.30 1.3 (Low Dose) 4 PRINT-Tre 680 4.44 133.5869.99 5.1 (Mid Dose) 5 PRINT-Tre 1370 8.94 266.6 1735.84 10.3 (HighDose) ^(a)= Based on the mean body weight of each group during thedosing period. ^(b)= Calculated with a content of 4% of Leucine in PRINTTreprostinil and PRINT Placebo and using Trehalose percentage of 93.5%in PRINT Placebo for Group 2 and Treprostinil percentage of 0.53% inPRINT Treprostinil for Groups 3 to 5.

The particle size MMADs from Groups 2 to 5 were between 1.3 and 2.0 μmgravimetrically (GSD 1.96 to 2.46); for both treprostinil and trehalose,chemical particle size distribution ranged from 1.3 to 2.1 μm with thecorresponding GSDs between 1.87 and 1.95. No mortality occurred. Noclinical signs were observed while coagulation, clinical chemistry andurinalysis parameters were unaffected and no test item-related findingswere seen macroscopically.

Rats tolerated daily administration of PRINT Placebo or PRINT-Tre at upto 1.37 mg/kg/day by 4-hour inhalation for 7 days.

Introduction

The objectives of the study were to:

-   -   1. Evaluate the toxicity of the test item, PRINT Treprostinil,        when administered to Sprague-Dawley rats by nose-only inhalation        for 4 hours a day for 7 days.    -   2. Evaluate the toxicity of the excipients that make up the        control item, PRINT Placebo, when administered to Sprague-Dawley        rats by nose-only inhalation for 4 hours a day for 7 days.    -   3. Determine the dose levels of PRINT Treprostinil for the        following 14-day GLP inhalation toxicology study from the        results of this dose range-finding study.

Experimental Design

Synopsis

The test and control items were administered to groups of 6 rats (3/sex)by 4-hour inhalation daily for 7 days, as described in the table below.The first day of dosing was designated as Day 1.

Targeted Aerosol Targeted Dose Level Leucine Group Concentration (ug/L)(mg/kg/day) Dose Level No. Test Material Treprostinil TrehaloseTreprostinil^(a) Trehalose^(b) (mg/kg/day)^(c) 1 Air Control 0 0 0 0 0 2PRINT Placebo^(b) 0 1684.6 0 262.9 11.2 3 PRINT-Tre (Low 1 175.2 0.1526.3 1.1 Dose) 4 PRINT-Tre (Mid 5 876.2 0.75 131.4 5.7 Dose) 5 PRINT-Tre(High 10 1752.5 1.5 262.9 11.3 Dose) ^(a)= Target aerosol concentrationswere calculated based on an estimated body weight of 0.250 kg ^(b)= Thetarget dose level for the placebo control was the same dose level as thehigh dose group (Group 5) ^(c)= Calculated with a content of 4% ofLeucine in PRINT Treprostinil and PRINT Placebo (using Trehalosepercentage of 93.5% in PRINT Placebo for Group 2 and Treprostinilpercentage of 0.53% in PRINT Treprostinil for Groups 3 to 5)

The high-dose level for PRINT-Tre was the feasible dose attainable basedon technical aerosol trials with the test item. (Study No. 41610).

The low- and mid-dose levels for PRINT-Tre were selected on the basis ofa previous PK study in rats (Study No. 75658).

Test and Control Item Information

Test Item Action

Treprostinil, the active ingredient in PRINT-Tre, is a prostacyclincompound approved for treatment of pulmonary arterial hypertension.

Characterization of Test Item

-   Content: 92.97% of Trehalose, 4% of Leucine, 2% of Tween80, 0.27% of    Sodium Citrate Dihydrate, 0.23% of Sodium Chloride: 0.53% of    Treprostinil sodium-   Storage Conditions: Cool (2 to 8° C.), protected from moisture    (e.g., desiccant)-   Handling Precautions: Standard laboratory precautions. Handled under    dry conditions (relative humidity ≤23%)-   Supplier: Liquidia Technologies Inc.

Characterization of Placebo Control Item

-   Content: -LKI-1R-983-3: 93.53% of Trehalose, 4% of Leucine, 2% of    Tween80, 0.24% of Sodium Citrate Dihydrate, 0.23% of Sodium Chloride    -   -LKI-1R-983-27: 93.5% of Trehalose, 4% of Leucine, 2% of        Tween80, 0.27% of Sodium Citrate Dihydrate, 0.23% of Sodium        Chloride-   Storage Conditions: Cool (2 to 8° C.), protected from moisture    (e.g., desiccant)-   Handling Precautions: Standard laboratory precautions. Handled under    dry conditions (relative humidity ≤23%)-   Supplier: Liquidia Technologies Inc.

Characterization of Air Control

Description: Medical Grade Air (NQ 5710-500/2000)

Supplied By: Kaeser SM-11 Air Compressor

Preparation of Test and Control Items

PRINT-Tre and PRINT Placebo were used as provided by the Sponsor. Aglove box under nitrogen was used for handling, aliquoting or packing ofthe canisters. Relative humidity (RH) inside the glove box was monitoredand recorded using a hygrometer and was kept below 23% RH.

Treatment

Acclimatization to Exposure System

Before the rats were presented to exposure atmosphere, rats wereaccustomed to the restraint procedure over a period of 3 days. Theanimals were gradually accustomed to restraint in the dosing tubes usedduring the exposures up to the duration that was used for aerosoladministrations.

Animal Exposure

-   Exposure system used: Flow-past rodent inhalation exposure system-   Exposure method: Inhalation by nose-only exposure-   Test and Control Item type: Air (Group 1), Dry Powder (Groups 2 to    5)-   Generation method: Piston feed/rotating brush generator (Groups 2 to    5)-   Duration of exposure: 240 minutes

The target aerosol concentrations and dose levels were as follows:

Targeted Targeted Targeted Targeted Estimated Total Inhaled AerosolTotal Inhaled Aerosol Total Inhaled Dose Level of Concentration DoseLevel of Concentration Dose Level of Group Group Treprostinil ofTreprostinil Trehalose of Trehalose Leucine No. Designation(mg/kg/day)^(a) (μg/L) (mg/kg/day)^(a) (μg/L) (mg/kg/day)^(c) 1 AirControl 0 0 0 0 0 2 Placebo Control^(b) 0 0 262.9 1684.6 11.2 3PRINT-Tre (Low 0.15 1 26.3 175.2 1.1 Dose) 4 PRINT-Tre (Mid 0.75 5 131.4876.2 5.7 Dose) 5 PRINT-Tre 1.5 10 262.9 1752.5 11.3 (High Dose) ^(a)=Target aerosol concentrations were calculated based on an estimated bodyweight of 0.250 kg ^(b)= The target dose level for the placebo controlwas the dose level as the high dose group (Group 5) ^(c)= Calculatedwith a content of 4% of Leucine in PRINT Treprostinil and PRINT Placebo(using Trehalose percentage of 93.5% in PRINT Placebo for Group 2 andTreprostinil percentage of 0.53% in PRINT Treprostinil for Groups 3 to5)

Estimation of Achieved Dose Levels

The target dose levels were estimated using the following formula:

$D_{L} = \frac{E_{c} \times {RMV} \times T}{BW}$

-   D_(L)=Achieved dose levels (mg/kg/day)=-   E_(c)=Actual concentration delivered to the animals (mg/L air)-   RMV=Respiratory Minute Volume (L/min) according to the method of    Bide, Armour and Yee. J. App. Toxicol., Vol. 20, 2000: RMV    (L/min)=0.499×BW (kg)°^(0.809)-   T=Time, duration of daily exposure (min.)-   BW=Mean body weight (kg) during exposure period.    This estimation of total inhaled dose assumed 100% deposition within    the respiratory tract.

Inhalation Exposure System

The powder aerosol for Groups 2 to 5 was produced using a pistonfeed/rotating brush generator. The aerosol produced was diluted asnecessary to achieve the target aerosol concentration and dischargedthrough a 40-mm diameter tube into a flow-past inhalation exposuresystem. The airflow rate through the exposure system was monitored andrecorded manually during each aerosol generation period. Airflow to theexposure system was controlled by the absolute volume of air supplyingthe generation apparatus using variable area flowmeters. Control of theaerosol exhaust flow from the animal exposure system was achieved usingan exhaust valve, and the overall balance of airflows in the exposuresystem was monitored using pressure gauges. The system provided aminimum of 1.0 L/min to each animal exposure port and was balanced toensure a slight positive pressure at the site of the animal exposure.This ensured that there was no dilution of the generated aerosol. Anequal delivery of aerosol to each exposure position was achieved byemploying a distribution network that was identical for each individualexposure position attached to the system.

Inhalation System Monitoring

Determinations of aerosol concentration, particle size distribution,oxygen concentration, relative humidity and temperature were measured onsamples collected from a representative port of the exposure chamber,with a collection sample flow-rate of 1 L/min. The sample flow rateswere precisely controlled using variable area flow meters that werecalibrated before use using a primary airflow calibrator. The absolutevolume of each aerosol concentration sample was measured with a wet typegas meter.

Oxygen Concentration

The oxygen concentration of the generated atmosphere was measured onceduring each aerosol exposure. Oxygen concentrations of the exposureatmospheres were maintained between 19-23%.

Relative Humidity/Temperature

The temperature and relative humidity of the generated atmosphere weremeasured once during each aerosol exposure. Temperatures of the exposureatmospheres were maintained between 19-24° C.

Determination of Aerosol Concentration

At least one aerosol concentration filter sample was collected for allgroups on each aerosol generation. The filter samples from Groups 3 to 5were weighed in order to measure the gravimetric concentration of thetest item in the generated aerosol. The filter samples were transferredto the analytical chemistry laboratory for chemical determination ofTreprostinil and Trehalose concentrations. The filter samples from Group2 were weighed in order to measure the gravimetric concentration of thecontrol item in the generated aerosol. The filter samples weretransferred to the analytical chemistry laboratory for chemicaldetermination of Trehalose concentration and to confirm the absence ofTreprostinil. The filter samples for Group 1 were not weighedgravimetrically and were only transferred to the analytical laboratoryto confirm the absence of Treprostinil and Trehalose. The analysis inthe analytical laboratory was performed using an analytical method(Study No. 41609 and 41635).

Determination of Aerosol Homogeneity

At least once during the study, atmosphere homogeneity in the exposuresystem was tested by collecting multiple aerosol samples from the top,middle and bottom tiers of the exposure system of Groups 2 to 5.

Determination of the Particle Size Distribution and Mass MedianAerodynamic Diameter (MMAD)

The distribution of particle size in the generated aerosols was measuredat least once for Groups 2 to 5 by collecting samples into a 7-StageMercer Cascade Impactor. All sample substrates obtained from Groups 3 to5 were weighed gravimetrically and then transferred to the analyticalchemistry laboratory for chemical determination of particle size ofaerosolized Treprostinil and Trehalose. All sample substrates obtainedfrom Group 2 were weighed gravimetrically and then transferred to theanalytical laboratory for determination of particle size of aerosolizedTrehalose. The analysis in the analytical laboratory was performed usingan analytical method (Study No. 41609 and 41635).

The MMAD and the Geometric Standard Deviation (GSD) were calculatedbased on the results obtained from the impactor using a log-probittransformation.

Reporting of Analytical Results

The analytical report containing the results from the filter andparticle size distribution sample analyses were prepared. Any samplesnot employed in the primary analysis or any remaining sample from theprimary analysis were retained until it was determined by the analystand Study Director that it was not required for confirmatory analysis.These samples were then discarded and their disposition was recorded inthe raw data.

Standard Operating Procedures

All procedures, were performed in accordance with the Standard OperatingProcedures and these were kept on file. Deviations to the StandardOperating Procedures were documented in the raw data.

Results

Inhalation System Monitoring

Oxygen Concentration, Temperature, and Relative Humidity

Exposure chamber conditions from the reported aerosol concentrationexposures are summarized below.

Humidity Temperature Oxygen Group (% RH) (° C.) Concentration (%) NumberAverage Average Average 1 28.6 20.4 20.9 2 39.6 21.2 20.9 3 35.6 21.620.9 4 26.2 21.1 20.9 5 31.1 20.8 20.9

Exposure atmosphere oxygen concentration, temperature and relativehumidity were considered acceptable throughout the study.

Aerosol Concentrations

Achieved gravimetric test atmosphere concentrations were as follows:

Targeted Aerosol Achieved Mean Aerosol Coefficient of GroupConcentration Concentration Variation % of No. (mg\L) (mg\L) (%) Target2 2.000* 1.966 34.7 98.3 3 0.200* 0.231 21.4 115.5 4 1.000* 0.928 20.592.8 5 2.000* 1.848 26.0 92.4 *Target aerosol concentrations were 0.140mg/L for Group 3, 0.700 mg/L for Group 4 and 1.400 mg/L for Groups 2 and5 for the first 2 days of exposure.

Achieved test atmosphere concentrations for treprostinil were asfollows:

Targeted Aerosol Achieved Mean Aerosol Coefficient of GroupConcentration Concentration Variation % of No. (μg\L) (μg\L) (%) Target3 1.0 1.10 22.1 110.0 4 5.0 4.44 21.0 88.8 5 10.0 8.94 28.1 89.4

Achieved test atmosphere concentrations for trehalose were as follows:

Targeted Aerosol Achieved Mean Aerosol Coefficient of GroupConcentration Concentration Variation % of No. (μg\L) (μg\L) (%) Target2 1684.6 1832.13 36.4 108.8 3 175.2 216.30 22.1 123.5 4 876.2 869.9921.2  99.3 5 1752.5 1735.84 29.4  99.0

The overall achieved aerosol concentrations for all groups were within20% of the targeted concentrations gravimetrically and for bothtreprostinil and trehalose, except for Group 3 for trehalose which was23.5% greater than the targeted concentration. The generated atmosphereswere considered stable over the treatment period even if all % CV wereall above 20% as this was due the wrong targeted gravimetricconcentrations being applied for the first 2 days of dosing. The overallaerosol concentrations were still considered acceptable for the study asthere was a significant difference in aerosol concentration betweengroups.

Aerosol Homogeneity

Achieved gravimetric test atmosphere homogeneity concentrations were asfollows:

Aerosol Aerosol Aerosol Concentration Concentration Concentration Groupof Top Tier of Middle Tier of Bottom Tier No. (mg/L) (mg/L) (mg/L) CV(%) 2 1.061 1.029 1.078 2.4 3 0.134 0.136 0.126 4.0 4 0.810 0.877 0.8454.0 5 1.225 1.263 1.280 2.2

Achieved test atmosphere homogeneity concentrations for treprostinilwere as follows:

Aerosol Aerosol Aerosol Concentration Concentration Concentration Groupof Top Tier of Middle Tier of Bottom Tier No. (μg/L) (μg/L) (μg/L) CV(%) 3 0.63 0.64 0.59 4.3 4 3.90 4.16 4.04 3.2 5 5.73 6.03 6.12 3.4

Achieved test atmosphere homogeneity concentrations for trehalose wereas follows:

Aerosol Aerosol Aerosol Concentration Concentration Concentration Groupof Top Tier of Middle Tier of Bottom Tier No. (μg/L) (μg/L) (μg/L) CV(%) 2 916.21 888.61 919.90 1.9 3 113.82 111.38 102.88 5.3 4 774.67887.67 780.97 7.8 5 1091.32  1153.45  1160.14  3.3

Chamber homogeneity of the aerosol concentrations were consideredacceptable since the coefficient of variance of aerosol concentrationbetween samples was not greater than 20%.

Particle Size Distribution

The average gravimetric particle size distribution measurement data wereas follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 2 89.1 81.8 51.3 19.3 11.6 9.4 7.9 0.0 2.0 2.46 78 3 97.9 95.0 79.635.5 21.1 15.1 5.7 0.0 1.3 1.96 94 4 95.5 89.4 60.3 24.1 13.4 9.0 4.80.0 1.7 2.07 88 5 96.9 92.3 63.1 28.2 15.7 8.5 4.8 0.0 1.6 1.99 91 MMAD= Mass median aerodynamic diameter GSD = Geometric standard deviation.

The chemical determinations of particle size distribution fortreprostinil were as follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 3 98.8 95.7 80.3 35.2 20.2 14.2 5.0 0.0 1.3 1.88 96 4 96.3 90.1 59.922.2 11.1 6.9 2.9 0.0 1.7 1.95 89 5 97.2 92.8 63.3 26.7 13.6 6.4 2.8 0.01.6 1.89 92 MMAD = Mass median aerodynamic diameter GSD = Geometricstandard deviation.

The chemical determinations of particle size distribution for trehalosewere as follows:

Cumulative % Less Than Stated Effective Mean % Group Cut-Off Diameter(μm) MMAD below 4 No. 4.60 3.00 2.10 1.60 1.10 0.70 0.33 0.00 (μm) GSDμm 2 89.3 85.2 52.1 17.7 8.1 4.0 0.0 0.0 2.1 1.74 87 3 96.6 93.3 77.431.0 15.4 12.0 3.4 0.0 1.5 1.94 93 4 97.3 90.9 59.3 19.9 8.0 5.3 2.7 0.01.8 1.88 90 5 97.2 94.4 62.6 25.0 12.1 5.6 2.8 0.0 1.6 1.87 92 MMAD =Mass median aerodynamic diameter GSD = Geometric standard deviation.

The particle size distribution was considered respirable for this studyas all MMADs were below 4 μm and the GSDs were within 1.5 and 3.

Estimation of Achieved Dose Levels

Overall achieved doses for treprostinil are presented below:

Targeted Duration of Body Estimated % of Group Dose Level ExposureWeight Achieved Doses Targeted No. (mg/kg/day) (min) Sex (kg)(mg/kg/day) Dose Level 3 0.15 240 Male 0.324 0.16 106.7  Female 0.2270.17 113.3  Combined 0.276 0.17 113.3  4 0.75 240 Male 0.316 0.66 88.0Female 0.229 0.70 93.3 Combined 0.273 0.68 90.7 5  1.5 240 Male 0.3151.33 88.7 Female 0.228 1.42 94.7 Combined 0.272 1.37 91.3

Overall achieved doses for trehalose are presented below:

Targeted Dose Duration of Body Estimated % of Group Level ExposureWeight Achieved Doses Targeted No. (mg/kg/day) (min) Sex (kg)(mg/kg/day) Dose Level 2 262.9 240 Male 0.316 273.4 104.0 Female 0.229290.8 110.6 Combined 0.273 281.2 107.0 3 26.3 240 Male 0.324 32.1 122.1Female 0.227 34.4 130.8 Combined 0.276 33.1 125.9 4 131.4 240 Male 0.316129.8 98.8 Female 0.229 138.1 105.1 Combined 0.273 133.5 101.6 5 262.9240 Male 0.315 259.2 98.6 Female 0.228 275.7 104.9 Combined 0.272 266.6101.4

Overall achieved doses for leucine are presented below:

Targeted Duration of Body Estimated % of Group Dose Level ExposureWeight Achieved Doses^(a) Targeted No. (mg/kg/day) (min) Sex (kg)(mg/kg/day) Dose Level 2 11.2 240 Male 0.316 11.7  104.5 Female 0.22912.4  110.7 Combined 0.273 12.0  107.1 3  1.1 240 Male 0.324 1.2 109.1Female 0.227 1.3 118.2 Combined 0.276 1.3 118.2 4  5.7 240 Male 0.3165.0  87.7 Female 0.229 5.3  93.0 Combined 0.273 5.1  89.5 5 11.3 240Male 0.315 10.0   88.5 Female 0.228 10.7   94.7 Combined 0.272 10.3  91.2 ^(a)Calculated with a content of 4% of Leucine in PRINT-Tre andPRINT Placebo (using Trehalose percentage of 93.5% in PRINT Placebo forGroup 2 and Treprostinil percentage of 0.53% in PRINT-Tre for Groups 3to 5)

Average achieved dose levels for all groups were within 20% of thetargeted dose levels, except for Group 3 for trehalose which was 26%above the targeted dose level; however, the dose levels were consideredacceptable for the study as a clear dose differentiation between groupsfor each sex was achieved.

Mortality

There were no mortalities during the study.

Clinical Signs

There were no clinical signs observed during the study.

Body Weight

The only differences in body weight or weight gain potentially relatedto administration of the test or control item were slightly less growth(weight gain) in males given PRINT-Tre at 0.68 mg/kg/day and in bothsexes given to PRINT-Tre at 1.37 mg/kg/day, relative to the air controlgroup. The pattern of differences implicates the active ingredienttreprostinil, not one of the excipients in PRINT-Tre.

These data are summarized in the table below, with differencespotentially related to treprostinil in bold.

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Males Starting weight (Day 1) (g) 301 309 315 312 312After 4 doses (Day 5) (g) 324 326 337 324 322 Change Absolute (g) +23+17 +22 +12 +10 Relative to Air Control — −6 −1 −11 −13 After 7 doses(fasted Day 8) 300 302 310 301 298 (g) Change Absolute (g)* −1 −7 −5 −10−14 Relative to Air Control — −6 −4 −9 −13 Females Starting weight(Day 1) (g) 220 223 220 227 226 After 4 doses (Day 5) (g) 231 235 232237 231 Change Absolute (g) +11 +12 +12 +10 +5 Relative to Air Control —+1 +1 −1 −6 After 7 doses (fasted Day 8) 205 209 204 213 211 (g) ChangeAbsolute (g)* −15 −14 −16 −14 −15 Relative to Air Control — +1 −1 −1 0*All animals were fasted overnight prior to necropsy.

Remaining differences were considered incidental and of no biologicalsignificance.

Hematology

The only differences in mean hematology parameters potentially relatedto administration of the test or control item were greater meanreticulocyte counts in all groups given PRINT-Tre, relative to the aircontrol group. The magnitude of difference was dose-related butstatistically significant only in males. The pattern of differencesimplicates the active ingredient treprostinil, not one of the excipientsin PRINT-Tre.

These data are summarized in the table below, with differencespotentially related to treprostinil in bold.

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) =Reticulocyte count 0 281 33 134 267 Males Mean (×10¹²/L) 0.226 0.2280.309 0.333 0.337 Relative to Air Control — +1% +37% +47% +49% FemalesMean (×10¹²/L) 0.179 0.176 0.214 0.290 0.283 Relative to Air Control —+1% +20% +62% +58%

An increase in reticulocyte count is an appropriate response to anincreased demand for RBCs. In this study, greater reticulocyte countswere not associated with differences in circulating erythron mass (i.e.,no differences in RBC count, haemoglobin concentration, or haematocrit).This suggests that the increased release of reticulocytes wasaccompanied by, and probably a response to, an increased rate of RBCloss, and that the erythropoietic response was adequate to maintainnormal circulating RBC numbers.

Remaining differences among mean hematology parameters were consideredincidental and of no biological significance.

Coagulation

There were no differences in mean coagulation parameters that wereconsidered to be related to administration of the test or control item.All differences were considered incidental and of no biologicalsignificance.

Clinical Chemistry

There were no differences in mean clinical chemistry parameters thatwere considered to be related to administration of the test or controlitem. All differences were considered incidental and of no biologicalsignificance.

Urinalysis

There were no differences in the urinalysis parameters that wereconsidered to be related to administration of the test or control item.All differences were considered incidental and of no biologicalsignificance.

Organ Weights

Differences in mean organ weight potentially related to administrationof the test or control item were noted for lungs, adrenal glands,thymus, and testes.

Remaining differences in mean organ weight were considered incidentaland of no biological significance.

Lungs

Mean lungs/trachea weights (absolute and relative to body weight) weregreater in all groups given the test or control item, compared to theair control group. The differences were greater with PRINT-Tre than withPRINT Placebo, and the differences were dose-related for PRINT-Tre. Thispattern suggests that administration of the excipients (likelytrehalose) resulted in a slight (15% to 17%) increase in lung weight,which was exacerbated by co-administration of treprostinil as the lungweights of PRINT-Tre groups were increased compared to the lung weightsof the PRINT Placebo group.

There was a histopathologic finding in the lungs that might haveaccounted for the greater lung weight; specifically, increased alveolarmacrophages with basophilic vacuolated cytoplasm in the lungs of allrats given PRINT Placebo or PRINT-Tre at ≥0.68 mg/kg/day. However,neither the distribution of this histopathologic finding across groupsnor the grade of the finding correlated well with the differences inmean lung weight, suggesting that some other factor was responsible.Because lungs were weighed before fixation, it is possible that somematerial responsible for the greater weight was removed during tissuefixation and processing.

Lung weight data are summarized in the table below, with differencespotentially related to PRINT Placebo and PRINT-Tre in bold.

Mean Lung Weight Data

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Males Absolute weight (g) 1.39 1.60 1.70 1.78* 1.90*Relative to Air Control — +15% +22% +28% +37% Relative weight 0.46 0.530.55 0.59* 0.64* (% body weight) Relative to Air Control — +15% +20%+28% +39% Females Absolute weight (g) 1.09 1.27 1.31* 1.48* 1.44*Relative to Air Control — +17% +20% +36% +32% Relative weight 0.53 0.610.64 0.70* 0.68* (% body weight) Relative to Air Control — +15% +21%+32% +28% *Statistically significant compared to air control; Dunnett's2-sided, p < 0.05

Thymus

Mean thymus weights (absolute and relative to body weight) were slightlylower in both sexes given PRINT-Tre at 0.68 mg/kg/day and in both sexesgiven to PRINT-Tre at 1.37 mg/kg/day, relative to the air control group(though not statistically significantly different). The pattern ofdifferences implicates the active ingredient treprostinil, not one ofthe excipients in PRINT-Tre as differences were also seen betweenPRINT-Tre groups and PRINT Placebo group. Lower thymus weight was notassociated with lower lymphocyte count or with any histopathologicfindings.

Lower thymus weight is one common manifestation of nonspecificphysiological or psychological stress (Everds et al., 2013). Becausethis finding was associated with reduced weight gain (growth) andsometimes also with greater adrenal glands weight, it was most likelysecondary to stress and not a direct effect of treprostinil.

Thymus weight data are summarized in the table below, with differencespotentially related to treprostinil in bold.

Mean Thymus Weight Data

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Males Absolute weight (mg) 504 593 537 388 429 Relativeto Air Control — +18% +7% −23% −15% Relative weight 0.168 0.198 0.1730.128 0.143 (% body weight) Relative to Air Control — +18% +3% −24% −15%Females Absolute weight (mg) 469 437 465 428 352 Relative to Air Control—  −7% −1%  −9% −25% Relative weight 0.229 0.210 0.228 0.202 0.166 (%body weight) Relative to Air Control —  −8% ±0% −12% −28%

Adrenal Glands

Mean adrenal glands weight (absolute and relative to body weight) wasgreater in males given PRINT-Tre at 0.17 mg/kg/day and in both sexesgiven to PRINT-Tre at 1.37 mg/kg/day, relative to the air control group(though not statistically significantly different). While thedifferences may have been due to chance and a consequence of the smallgroup sizes (3/sex), the pattern of differences raises the possibilitythat they are related to administration of treprostinil, at least at thehigh-dose level as differences were also seen between the high dosePRINT-Tre group and the PRINT Placebo group. Greater adrenal glandsweight was not associated with any histopathologic findings.

Greater adrenal glands weight is one common manifestation of nonspecificphysiological or psychological stress (Everds et al., 2013). Becausethis finding was associated with reduced weight gain (growth) and lowerthymus weight at the high-dose level, it was most likely secondary tostress and not a direct effect of treprostinil.

Adrenal glands weight data are summarized in the table below, withdifferences potentially related to treprostinil in bold.

Mean Adrenal Glands Weight Data

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Males Absolute weight (mg) 62 71 76 68 78 Relative to AirControl — +15% +23% +10% +26% Relative weight 0.021 0.023 0.025 0.0230.027 (% body weight) Relative to Air Control — +10% +19% +10% +29%Females Absolute weight (mg) 74 73 70 76 89 Relative to Air Control — −1%  −5%  +3% +20% Relative weight 0.036 0.035 0.035 0.036 0.042 (%body weight) Relative to Air Control —  −3%  −3%  ±0% +17%

Testes

There was a trend toward slightly lower mean testes weight (absolute andrelative to body weight) in groups given PRINT-Tre at ≥0.68 mg/kg/day,relative to the air control group. While the differences may have beendue to chance and a consequence of the small group sizes (3/sex), thepattern of differences raises the possibility that they are related toadministration of treprostinil as differences were also seen between themid and high dose PRINT-Tre groups and the PRINT Placebo group. Slightlylower testes weight was not associated with any histopathologicfindings.

Testes weight data are summarized in the table below, with differencespotentially related to treprostinil in bold.

Mean Testes Weight Data

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Absolute weight (g) 3.51 3.40 3.39 3.25 3.15 Relative toAir Control — −3% −3% −7% −10% Relative weight 1.17 1.13 1.10 1.08 1.06(% body weight) Relative to Air Control — −3% −6% −8%  −9%

Macroscopic Findings

There was no evidence of test item-related macroscopic findings atnecropsy.

All findings were considered to be incidental as they were notdose-related, of low incidence, or occurred in the air control, placebocontrol and treated animals.

Microscopic Findings

Treatment-related findings were observed in the lungs, anterior nasalcavity, and nasopharynx. All other microscopic findings were consideredto be incidental or procedure-related.

Lungs

In the lungs, minimal to mild increased alveolar macrophages withbasophilic vacuolated cytoplasm were observed in all rats given PRINTPlacebo or PRINT-Tre at ≥0.68 mg/kg/day. The pattern of this findingacross groups indicates that it is a response to the excipients (likelytrehalose). There were no associated inflammatory changes in the lungs.Increased alveolar macrophages are a common finding in inhalationtoxicity studies with powders. It reflects normal pulmonary clearance ofinhaled particles and is not considered to be adverse.

These data are summarized in the table below, with differencespotentially related to test or control item in bold.

Incidence and Grade of Increased Alveolar Macrophages

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Males Incidence 0/3 3/3 0/3 3/3 3/3 Mean grade — 1.7 —1.0 1.7 Females Incidence 0/3 3/3 0/3 3/3 3/3 Mean grade — 2.0 — 1.0 1.7

Nasal Cavity and Nasopharynx

Goblet-cell hypertrophy/hyperplasia was seen in the cranial portion ofthe nasal cavity and in the nasopharynx of at least one rat in allgroups given PRINT Placebo or PRINT-Tre, but the incidence was greaterin groups given PRINT-Tre at ≥0.68 mg/kg/day, and the mean grade wasgreater in the group given PRINT-Tre at 1.37 mg/kg/day. This patternsuggests that administration of the excipients (likely trehalose)resulted in occasional goblet-cell changes, which were exacerbated byco-administration of treprostinil at higher dose levels.

Goblet cell hypertrophy/hyperplasia in the anterior nasal cavity andnasopharynx is one of the most frequently observed lesions in rodentsexposed to irritant compounds. This finding generally is considered anonspecific protective or adaptive response and not adverse.

These data are summarized in the table below, with differencespotentially related to test or control item in bold.

Incidence and Grade of Goblet-Cell Hypertrophy/Hyperplasia

Test Material = Air PRINT Placebo PRINT-Tre Treprostinial Dose Level(mg/kg/day) = 0 0 0.17 0.68 1.37 Trehalose Dose Level (mg/kg/day) = 0281 33 134 267 Nasal Cavity Males Incidence 0/3 1/3 1/3 3/3 3/3 Meangrade — 1.0 1.0 1.0 1.3 Females Incidence 0/3 0/3 0/3 1/3 2/3 Mean grade— — — 1.0 1.5 Nasopharynx Males Incidence (all graded minimal) 0/3 0/30/3 3/3 3/3 Females Incidence(all graded minimal) 0/3 1/3 0/3 3/3 3/3

Discussion and Conclusions

Rats tolerated daily administration of PRINT Placebo or PRINT-Tre at upto 1.37 mg/kg/day by 4-hour inhalation for 7 days.

The only findings potentially related to administration of excipients(likely trehalose) were:

-   -   Increased alveolar macrophages with basophilic vacuolated        cytoplasm in all rats given PRINT Placebo or PRINT-Tre at ≥0.68        mg/kg/day; i.e., in rats given trehalose at ≥134 mg/kg/day. The        mean grade of this finding increased with trehalose dose level.        This finding was not associated with inflammatory changes in the        lungs and was considered to reflect normal pulmonary clearance        of inhaled particles. It was not considered to be adverse.    -   Greater mean lung weight in groups given PRINT Placebo or        PRINT-Tre. The weight differences were unrelated to trehalose        dose level. Instead, they were greater with PRINT-Tre than with        PRINT Placebo and were dose-related for PRINT-Tre. This pattern        suggests that administration of the excipients (likely        trehalose) resulted in a slight (15% to 17%) increase in lung        weight, which was exacerbated by co-administration of        treprostinil. Of note, the pattern of differences in lung weight        across groups is distinct from the pattern of increased alveolar        macrophages across groups, indicating that the weight        differences were not a consequence of increased macrophages.        There were no histopathologic findings in the lungs that might        have accounted for the greater lung weight. Because lungs were        weighed before fixation, it is possible that some material        responsible for the greater weight was removed during tissue        fixation and processing.    -   Minimal goblet-cell hypertrophy/hyperplasia in the cranial        portion of the nasal cavity of at least one rat in all groups        given PRINT Placebo or PRINT-Tre. The incidence of this finding        was unrelated to trehalose dose level. Instead, the incidence        was greater with PRINT-Tre at ≥0.68 mg/kg/day, and the mean        grade was greater with PRINT-Tre at 1.37 mg/kg/day. This pattern        suggests that administration of the excipients (likely        trehalose) resulted in occasional goblet-cell changes, which        were exacerbated by co-administration of treprostinil at higher        dose levels. Goblet-cell hypertrophy/hyperplasia was considered        a nonspecific protective or adaptive response and not adverse.

Besides exacerbating lung weight differences and goblet-cellhypertrophy/hyperplasia in the nasal cavity and nasopharynx, thefollowing other findings were potentially related to administration oftreprostinil as PRINT-Tre:

-   -   Slightly less growth (weight gain) in males at 0.68 mg/kg/day        and in both sexes at 1.37 mg/kg/day.    -   Greater mean reticulocyte counts at all dose levels, with the        magnitude of difference increasing with dose level. This was not        considered adverse in and of itself; however, it likely        reflected an appropriate adaptive response to an increased rate        of RBC loss or turnover.    -   Greater mean adrenal glands weight in males at 0.17 mg/kg/day,        lower mean thymus weight in both sexes at 0.68 mg/kg/day, and        greater mean adrenal glands weight and lower mean thymus weight        in both sexes at 1.37 mg/kg/day. There were no associated        differences in lymphocyte count or histopathologic findings in        either organ. These organ weight differences most likely        reflected stress and were not a direct effect of treprostinil.

Based on these results, it is recommended that an upcoming 14-day GLPinhalation toxicology study in rats target similar dose levels as usedin the current study.

Clinical Study: LIQ861

Randomized, Placebo-Controlled, Single-Ascending Dose Study EvaluatingPharmacokinetics (PK) and Safety in Healthy Male and Female Volunteers

A clinical study was conducted to (1) determine the single-dose safetyand tolerability and (2) evaluate the single-dose pharmacokinetics ofparticles of the invention upon administration to healthy male andfemale subjects.

Six cohorts were evaluated: dose levels of 25, 50, 75, 100, 125 and 150μg of treprostinil respectively. In each cohort, eight subjects wererandomly assigned in a 3:1 blinded ratio and received a single dose ofeither particles of the invention (N=6) or placebo particles (N=2).

Blood was collected for PK evaluation at T=0, 5, 10, 15, 20, 25, 30, 45,60, 90, and 120 minutes and 3, 4, 6, and 8 hours post-dose.

Cohort 1

Eight subjects were enrolled and dosed in Cohort 1. Six subjectsreceived active treatment and 2 received placebo. Active treatment wasadministered by dry powder inhalation (DPI) as a single capsule of 25 μgtreprostinil strength, and placebo treatments were administered by DPIas a single capsule of the placebo formulation. All inhalations wereadministered using the RS00 inhaler.

Blood samples were obtained prior to dosing and at nominal times of0.083, 0.167, 0.25, 0.33, 0.417, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4,6, and 8 hours after inhalation. Analysis of plasma concentration versustime data for calculation of standard pharmacokinetic (PK) parametersfollowing inhalation was conducted using Phoenix WinNonlin version 6.3using scheduled blood sampling times. Plasma concentrations wereprovided only for subjects on active dose and random subject numberswere assigned by the bioanalytical laboratory to retain the study blind.

The table shown in FIG. 3A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 1. Preliminarynon-compartmental PK parameters for treprostinil are summarized in thetable shown in FIG. 3B. The highest concentrations for three of the sixsubjects occurred at 0.33 hours post-inhalation; one subject each had aTmax of 0.167, 0.25, and 0.417 hours post-dose. Concentrationssubsequently decayed with a single-phase disposition profile, as shownin the log-linear plots. At two hours after inhalation, two of sixactive subjects had measurable concentrations of treprostinil and onlyone subject had measurable concentrations at 2.5 and 3 hours afterinhalation. No subjects had quantifiable concentrations after the 3 hourtimepoint.

The Cmax averaged 0.364 ng/mL and the most frequent Tmax was 0.33 hoursafter inhalation. AUCinf values averaged 0.301 h*ng/mL with a CV % of30.2%. The apparent volume of distribution (Vz/F) averaged 68.1 L. Oralclearance (CL/F) averaged 91.0 L/h and ranged from 59.1 to 150.Variability in the CL/F value had a CV % of 35.8%.

Cohort 2

Nine subjects were enrolled and dosed in Cohort 2. At least six subjectsreceived active treatment and at least 2 received placebo; 1 subjectwithdrew before the 2 hour PK sample and was replaced. Subjects withtruncated sampling schedules have been excluded in this interimanalysis. Active treatment was administered by dry powder inhalation(DPI) as a single capsule of 50 μg treprostinil strength, and placebotreatments were administered by DPI as a single capsule of the placeboformulation. All inhalations were administered using the RS00 inhaler.

Blood samples were obtained prior to dosing and at nominal times of0.083, 0.167, 0.25, 0.33, 0.417, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4,6, and 8 hours after inhalation. Analysis of plasma concentration versustime data for calculation of standard pharmacokinetic (PK) parametersfollowing inhalation was conducted using Phoenix WinNonlin version 6.3using scheduled blood sampling times. Plasma concentrations wereprovided only for subjects on active dose and random subject numberswere assigned by the bioanalytical laboratory to retain the study blind.

The table shown in FIG. 4A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 2.

The highest concentrations for four of the six subjects occurred at 0.17hours post-inhalation; of the remaining subjects, one had Tmax at 0.083hours post-dose and one at 0.417 hours post-dose. At 2.5 hours afterinhalation, 2 of 6 active subjects had measurable concentrations oftreprostinil and only one subject had measurable concentrations at 3hours after inhalation. No subjects had quantifiable concentrationsafter the 3 hour timepoint.

Preliminary non-compartmental PK parameters for treprostinil for Cohort2 are summarized in the table shown in FIG. 4B. The Cmax averaged 0.572ng/mL and the most frequent Tmax was 0.167 hours after inhalation.AUCinf values averaged 0.422 h*ng/mL with a CV % of 62.8%. The apparentvolume of distribution (Vz/F) averaged 110 L. Oral clearance (CL/F)averaged 208 L/h and ranged from 67 to 624. Variability in the CL/Fvalue had a CV % of 101.5%.

By comparison, the Cmax for Cohort 1 averaged 0.364 ng/mL and the AUCinfvalues averaged 0.301 h*ng/mL. Thus, a doubling of the treprostinil doseresulted in an approximate 50% increase in exposure. The Vz/F and theCL/F values were considerably higher for Cohort 2 and with greatervariability.

Cohort 3

Eight subjects were enrolled and dosed in Cohort 3. Six subjectsreceived active treatment and two received placebo. Active treatment wasadministered by dry powder inhalation (DPI) as a single capsule of 75 μgtreprostinil strength and placebo treatments were administered by DPI asa single capsule of the placebo formulation. All inhalations wereadministered using the RS00 inhaler.

Blood samples were obtained prior to dosing and at nominal times of0.083, 0.167, 0.25, 0.33, 0.417, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4,6, and 8 hours after inhalation. Analysis of plasma concentration versustime data for calculation of standard pharmacokinetic (PK) parametersfollowing inhalation was conducted using Phoenix WinNonlin version 6.3using scheduled blood sampling times. Plasma concentrations wereprovided only for subjects on active dose and random subject numberswere assigned by the bioanalytical laboratory to retain the study blind.

The table shown in FIG. 5A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the 6 active subjects in Cohort 3.

The highest concentrations for three of the six subjects occurred at0.25 hours post-inhalation; of the remaining subjects, 1 had Tmax at0.083 hours post-dose, 1 at 0.17 hours post-dose, and 1 at 0.417 hourspost-dose. At 3 hours after inhalation, two of six active subjects hadmeasurable concentrations of treprostinil. No subjects had quantifiableconcentrations after the 3 hour timepoint.

Preliminary non-compartmental PK parameters for treprostinil for Cohort3 are summarized in the table shown in FIG. 5B. The Cmax averaged 0.728ng/mL and the most frequent Tmax was 0.25 hours after inhalation. AUCinfvalues averaged 0.757 h*ng/mL with a CV % of 39.4%. The apparent volumeof distribution (Vz/F) averaged 97 L. Oral clearance (CL/F) averaged 112L/h and ranged from 58 to 161. Variability in the CL/F value had a CV %of 39.4%.

By comparison, the Cmax for Cohort 1 and Cohort 2 averaged 0.364 ng/mLand 572 ng/mL, respectively, while the AUCinf values averaged 0.301h*ng/mL and 0.422 h*ng/mL. Thus, a tripling of the dose from Cohort 1resulted in an approximate 100-150% increase in exposure. The CL/Fvalues were for Cohort 3 were more consistent with Cohort 1, and withsimilar variability, than what was observed in Cohort 2. The resultsindicate that both Cmax and AUCinf may be increasing proportionately tothe increase in the dose and that the CL is independent of dose over therange of 25 to 75 μg treprostinil.

Cohort 4

Eight subjects were enrolled and dosed in Cohort 4. Six subjectsreceived active treatment and two received placebo. Active treatment of100 μg treprostinil was administered by dry powder inhalation (DPI) as 2capsules of 50 μg treprostinil strength and placebo treatments wereadministered by DPI as 2 capsules of the placebo formulation. Allinhalations were administered using the RS00 inhaler.

Blood samples were obtained prior to dosing and at nominal times of0.083, 0.167, 0.25, 0.33, 0.417, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4,6, and 8 hours after inhalation. Analysis of plasma concentration versustime data for calculation of standard pharmacokinetic (PK) parametersfollowing inhalation was conducted using Phoenix WinNonlin version 6.3using scheduled blood sampling times. Plasma concentrations wereprovided only for subjects on active dose and random subject numberswere assigned by the bioanalytical laboratory to retain the study blind.

The table shown in FIG. 6A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 4. The highestconcentrations for 2 of the 6 subjects occurred at 0.25 hourspost-inhalation; of the remaining subjects, 2 had Tmax at 0.5 hourspost-dose, 1 at 0.17 hours post-dose, and 1 at 0.33 hours post-dose. At4 hours after inhalation, 3 of 6 active subjects had measurableconcentrations of treprostinil. No subjects had quantifiableconcentrations at the 6 or 8 hour timepoints.

Preliminary non-compartmental PK parameters for treprostinil for Cohort4 are summarized in FIG. 6B. The Cmax averaged 1.08 ng/mL and the mostfrequent Tmax values were observed at 0.25 hours and 0.5 hours afterinhalation. AUCinf values averaged 1.22 h*ng/mL with a CV % of 18.4%.The apparent volume of distribution (Vz/F) averaged 96 L. Oral clearance(CL/F) averaged 84.8 L/h and ranged from 68.3 to 122. Variability (CV %)in the CL/F value was 22.8%.

By comparison, the Cmax for Cohorts 1, 2, and 3 averaged 0.364 ng/mL,0.572 ng/mL, and 0.728 ng/mL respectively, while the AUCinf valuesaveraged 0.301 h*ng/mL, 0.422 h*ng/mL, and 0.757 h*ng/mL. Thus, aquadrupling of the dose from Cohort 1 resulted in an approximate200-300% increase in exposure, while a doubling of the dose from Cohort2 resulted in an approximate 2-fold increase in exposure. The resultsindicate that both Cmax and AUCinf may be increasing proportionately tothe increase in the dose and that the CL/F is independent of dose overthe range of 25 to 100 μg treprostinil.

Cohort 5

Eight subjects were enrolled and dosed in Cohort 5. Six subjectsreceived active treatment and two received placebo. Active treatment of125 μg treprostinil was administered by dry powder inhalation (DPI) as 1capsule of 75 μg and 1 capsule of 50 μg treprostinil strength andplacebo treatments were administered by DPI as 2 capsules of the placeboformulation. All inhalations were administered using the RS00 inhaler.

Blood samples were obtained prior to dosing and at nominal times of0.083, 0.167, 0.25, 0.33, 0.417, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4,6, and 8 hours after inhalation. Analysis of plasma concentration versustime data for calculation of standard pharmacokinetic (PK) parametersfollowing inhalation was conducted using Phoenix WinNonlin version 6.3using scheduled blood sampling times. Plasma concentrations wereprovided only for subjects on active dose and random subject numberswere assigned by the bioanalytical laboratory to retain the study blind.

The table shown in FIG. 7A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the 6 active subjects in Cohort 5. The highestconcentrations for 3 of the 6 subjects occurred at 0.17 hourspost-inhalation; of the remaining subjects, 2 had Tmax at 0.33 hourspost-dose, and 1 at 0.42 hours post-dose. At 3.5 and 4 hours afterinhalation, only 1 of 6 active subjects had measurable concentrations oftreprostinil. No subjects had quantifiable concentrations at the 6 or 8hour timepoints.

Preliminary non-compartmental PK parameters for treprostinil for Cohort5 are summarized in FIG. 7B. The Cmax averaged 1.19 ng/mL and the mostfrequent Tmax values were observed at 0.17 hours after inhalation.AUCinf values averaged 1.15 h*ng/mL with a CV % of 44.9%. The apparentvolume of distribution (Vz/F) averaged 101 L. Oral clearance (CL/F)averaged 141 L/h and ranged from 65.7 to 336. Variability (CV %) in theCL/F value was 69.9%.

By comparison, the Cmax for Cohorts 1, 2, 3, and 4 averaged 0.364 ng/mL,0.572 ng/mL, 0.728 ng/mL, and 1.08 ng/mL respectively, while the AUCinfvalues averaged 0.301 h*ng/mL, 0.422 h*ng/mL, 0.757 h*ng/mL, and 1.22h*ng/mL. Thus, a quintupling of the dose from Cohort 1 resulted in anapproximate 220-280% increase in exposure. The results indicate thatboth Cmax and AUCinf may be increasing proportionately to the increasein the dose and that the CL/F is independent of dose over the range of25 to 125 μg treprostinil.

Cohort 6

Cohort 6 was conducted as an original and a repeat. In each Cohort 6(original and repeat), eight subjects were enrolled and dosed. Sixsubjects received active treatment and two received placebo. Activetreatment of 150 μg treprostinil was administered by dry powderinhalation (DPI) as 2 capsules of 75 μg treprostinil strength andplacebo treatments were administered by DPI as 2 capsules of the placeboformulation. All inhalations were administered using the RS00 inhaler.Cohort 6 original included some mechanical device failures and subjectnon-compliance with instructions, giving rise to Cohort 6 repeat.

Blood samples were obtained prior to dosing and at nominal times of0.083, 0.167, 0.25, 0.33, 0.417, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4,6, and 8 hours after inhalation. Analysis of plasma concentration versustime data for calculation of standard pharmacokinetic (PK) parametersfollowing inhalation was conducted using Phoenix WinNonlin version 6.3using scheduled blood sampling times. Plasma concentrations wereprovided only for subjects on active dose and random subject numberswere assigned by the bioanalytical laboratory to retain the study blind.

The table shown in FIG. 8A contains a summary of the treprostinilconcentration-time data for individual subjects with descriptivestatistics for the six active subjects in Cohort 6-R. The highestconcentrations for 2 of the 6 subjects occurred at 0.25 hourspost-inhalation and at 0.33 hours post-inhalation. In the remaining 2subjects, Tmax occurred at the 0.167 and 0.417 hours post-dosetimepoints. At 4 hours after inhalation, 4 of 6 active subjects hadmeasurable concentrations of treprostinil. No subjects had quantifiableconcentrations at the 6 or 8 hour timepoints.

Preliminary non-compartmental PK parameters for treprostinil for Cohort6-R are summarized in FIG. 8B. Preliminary non-compartmental PKparameters for treprostinil for Cohort 6-Original are summarized in FIG.8C. Mean concentration-time data for each of the six cohorts isdisplayed on a linear scale in FIG. 8D. The Cmax averaged 1.45 ng/mL andthe most frequent Tmax values were observed at 0.25 and 0.33 hours afterinhalation. AUCinf values averaged 1.62 h*ng/mL with a CV % of 68.3%.The apparent volume of distribution (Vz/F) averaged 107 L. Oralclearance (CL/F) averaged 126 L/h and ranged from 51.8 to 245.Variability (CV %) in the CL/F value was 68.3%.

By comparison, the Cmax for Cohorts 1, 2, 3, 4, 5, and the combinedCohort 6 averaged 0.364 ng/mL, 0.572 ng/mL, 0.728 ng/mL, 1.08 ng/mL,1.19 ng/mL, and 1.21 ng/mL, respectively (FIG. 8E), while the AUCinfvalues averaged 0.301 h*ng/mL, 0.422 h*ng/mL, 0.757 h*ng/mL, 1.22h*ng/mL, 1.15 h*ng/mL, and 1.37 h*ng/mL (FIG. 8F). Thus, a 6-foldincrease of the dose from Cohort 1 to the combined Cohort 6 observationsresulted in an approximate 260-400% increase in exposure, while triplingfrom Cohort 2 and doubling from Cohort 3 resulted in approximateincreases of exposure by 130-255% and 81-98%, respectively. Moreover,plots of the relationship between dose and Cmax and AUCinf are displayedin FIG. 8E and FIG. 8F, respectively. The results indicate that bothCmax and AUCinf may be increasing proportionately to the increase in thedose. It was observed at the CRU during the original 150 μg dosing,however, that there were several apparent device failures that may haveresulted in incomplete and/or inefficient exposures. It should be notedthat no device failures were noted during the repeat dosing and, whilemean values may be higher than in the initial cohort, the variability inthe repeated cohort is greater. A plot of the relationship between doseand CL/F (FIG. 8G) shows that the CL/F is independent of dose over therange of 25 to 150 μg treprostinil, which suggests that PK oftreprostinil has a proportional relationship to dose over the range of25 to 150 μg treprostinil.

Clinical Conclusions

LIQ861 was dosed at levels of 25, 50, 75, 100, 125 and 150 μgtreprostinil from either a single capsule (25, 50 and 75 mcg doses) or acombination of two lower capsular strengths (for 100, 125 and 150 mcgdoses), each capsule either undergoing either a single breath or twobreaths. According to embodiments of the present invention, novelcapsule to particle powder to active ingredient ratios, and breath percapsule and powder per breath ratios for human dosing are included inthe following table.

Patient presentation of particle powder and active agent per capsule perbreath for particle formulation having 0.5 percent active agent loadCapsules 1 1 1 2 2 2 Particle  5 10 15 Combination Combination of,Combination of, Powder in of two 50 ex., 1 at 50 mcg ex., two capsulesmg mcg and 1 at 75 mcg at 75 mcg capsules or one 25 mcg and one 75 mcgActive 25 50 75 Varies, see Varies, see above Varies, see above AgentLoad above in mcg Breaths to 1 to 2 1 to 2 1 to 2 1 to 2 per 1 to 2 percapsule 1 to 2 per capsule Deliver capsule Particle 2.5-5  5-10  7.5-15Varies, up to Varies, up to 15 Varies, up to 15 Powder per 15 Breath inmg Active 12.5-25 25-50 37.5-75 Varies, up to Varies, up to 75 Varies,up to 75 Agent per 75 Breath in mcg

According to such embodiments, as shown in the above table, each breathcan receive from 2.5-15 mg of particle power and from 12.5-75 mcg ofactive agent.

For the given treprostinil delivered in the given mass of particlepowder loaded into a capsule and delivered through a dry powder inhalerresults in the human clinical outcomes are included in the followingtable for LIQ861.

LIQ861 Clinical C_(max) T_(max) ^(a) t_(1/2) AUC_(last) AUC_(inf) CL/FVz/F Outcomes (ng/mL) (h) (h) (h * ng/mL) (h * ng/mL) (L/h) (L) 25 mcg0.36 0.33 0.52 0.27 0.3 91 68.1 Treprostinil (0.12) (0.17, 0.42) (0.16)(0.09) (0.09) (32.6) (27.4) 50 mcg 0.57 0.17 0.45 0.4 0.42 208 110Treprostinil (0.37) (0.08, 0.42) (0.12) (0.26) (0.27) (211) (66.6) 75mcg 0.73 0.25 0.62 0.72 0.76 112 97 Treprostinil (0.3) (0.08, 0.42)(0.18) (0.31) (0.31) (38.5) (29.1) 100 mcg 1.08 0.29 0.78 1.18 1.22 84.895.5 Treprostinil (0.31) (0.17, 0.5) (0.13) (0.22) (0.23) (19.3) (28.2)125 mcg 1.19 0.25 0.53 1.12 1.15 141 101 Treprostinil (0.53) (0.17,0.42) (0.07) (0.51) (0.52) (98.8) (58.7) 150 mcg 1.21 0.29 0.66 1.331.37 119 115 Treprostinil (0.3) (0.08, 0.42) (0.15) (0.44) (0.42) (35.8)(51.4) 150 mcg 1.45 0.29 0.64 1.58 1.62 126 107 Treprostinil (0.63)(0.17, 0.42) (0.11) (0.85) (0.87) (80.3) (54) Abbreviations: SD =standard deviation; C_(max) = maximum observed plasma concentration;T_(max) = time to C_(max); t_(1/2) = half-life; AUC = area under thecurve; CL/F = apparent clearance; Vz/F = apparent volume of distributionAll values except for T_(max) are reported as arithmetic means with SDin parentheses. ^(a)T_(max) reports median values with minimum andmaximum values in parentheses

For comparison, TYVASO (United Therapeutics, Inc.) provides the currentstandard of treatment for inhaled treprostinil. Such treprostinil isdelivered through a nebulizer for the treatment of PAH and is limited todeliver 6 mcg of treprostinil per breath, utilizing 9 breaths to reach a54 mcg dose. The current standard of inhaled treatment has shown to bedose limited to a maximum tolerated dose of 84 mcg of treprostinil,which required 14 breaths to reach such dose. See, Channick, R. et al.,Inhaled Treprostinil: a therapeutic review, Drug Design, Development andTherapy 2012:6 19-28; and Nelsen A C, et al., Pharmacokinetics OfInhaled Treprostinil Sodium In Healthy Volunteers. Am J Respir Crit CareMed. 2010; 181:A3348; both of which are incorporated herein by referencein their entirety.

In alternative embodiments, particles of the present invention mayinclude 1% treprostinil load, as compared to 0.5% treprostinil load ofthe LIQ861 particles. According to an embodiment of the presentinvention, a plurality of 1% treprostinil particles were fabricated froma solution comprising, weight percent solids in water, of: 1.06%treprostinil sodium, 92.44% trehalose dihydrate, 2% polysorbate 80, 4% Lleucine, 0.27% sodium citrate dihydrate, and 0.23% sodium chloride.

According to a 1 percent treprostinil particle formulation of thepresent invention, particle powder mass and active agent presented to apatient comprise the following novel capsule to particle powder toactive ingredient ratios, and breath per capsule and powder per breathratios for human dosing.

Patient presentation of particle powder and active agent per capsule perbreath for particle formulation having 1 percent active agent loadCapsules 1 1 1 1 1 1 Particle Powder in mg 2.5 5 7.5 10 12.5 15 ActiveAgent 25 50 75 100 125 150 Load in mcg Breaths to 1 to 2 1 to 2 1 to 2 1to 2 1 to 2 1 to 2 Deliver Particle Powder  1.25-2.5 2.5-5    3.75-7.55-10  6.25-12.5 7.5-15  per Breath in mg Active Agent per 12.5-25 25-5037.5-75 50-100 62.5-125  75-150 Breath in mcg

According to such embodiments, as shown in the above table, each breathcan receive from 1.25-15 mg of particle power and from 12.5-150 mcg ofactive agent.

For the powder mass found acceptable in LIQ861 initial clinical trialassociated with delivery of the 150 mcg dose, at a 1% active drugparticle a dose of 300 mcg of active drug can be administered in a safeand acceptable powder mass and excipient quantity.

Kits

According to embodiments of the present invention the dry powder inhalerdevice can be combined into a kit with capsules for use therein. Thecapsules can be packaged in blister packs with or without desiccant toensure controlled environment for the LIQ861 particle powder while thetraveling with a user. The blister packs can include capsules for asingle dosing or multiple capsules for a day, week or month of doses.Typically a patient will treat 4 times per day for the PAH indication.The kit can include capsules comprising dosage strengths of 25, 50, 75,100, 125, 150, 200, 250, 300 mcg or beyond for the treatment of PAH. Theparticles of the powder in the capsules of the kits can be particlescomprising 0.5% treprostinil or 1% treprostinil.

Abbreviations and Nomenclature Cross-references 6MWD 6 Minute WalkDistance AE Adverse Event AUC Area Under the Curve AUCinf Area Under theConcentration-Time Curve Extrapolated to Time Infinity AUClast AreaUnder the Concentration-Time Curve to the Last Measured Timepoint AUCextPercentage of Area Under the Curve Extrapolated Beyond Last MeasureableConcentration AVT Acute Pulmonary Vasodilator Testing BA BioavailabilityBDI Borg Dyspnea Index BLQ Below the Limit of Quantitation BMPR2 BoneMorphogenic Protein Receptor Type II Gene BP British Pharmacopoeia BTOBenzidine Triol CAS Chemical Abstracts Service CFR Code of FederalRegulations CFU Colony Forming Unit cGMP Current Good ManufacturingPractice CI Cardiac Index CL Clearance Cmax Maximum Concentration CMCChemistry Manufacturing and Controls CO Cardiac Output COPD ChronicObstructive Pulmonary Disease Css Concentration at Steady State CTEPHChronic Thromboembolic Pulmonary Hypertension CYP Cytochrome P450 DMFDrug Master File DP Drug Product DPI Dry Powder Inhaler DPPADiphenylphosphinic Acid DRF Dose Range Finding DS Drug Substance DUSADosage Unit Sampling Apparatus eCMH Extended Cochran-Mantel-HaenszelTest ECG Electrocardiogram ED Emitted Dose Emax Maximum Effect EPEuropean Pharmacopoeia ERA Endothelin Receptor Agonists ET3 PolyethyleneTerephthalate Cyclic Trimer EU Endotoxin Unit ESRD End-Stage RenalDisease F Bioavailability FCR Fluorocur ® FDA Food and DrugAdministration FPF Fine Particle Fraction Frel Relative BioavailabilityFT-IR Fourier Transform Infrared Spectroscopy GC Gas Chromatography GLPGood Laboratory Practice GMP Good Manufacturing Practice GSD GeometricStandard Deviation HIAC High Accuracy Particle Counter HIV HumanImmunodeficiency Virus H-L Hodges-Lehmann HPLC High Performance LiquidChromatography HPMC Hydroxypropyl Methylcellulose HR Heart Rate ICHInternational Conference on Harmonisation IM Intramuscular INDInvestigational New Drug INR International Normalized Ratio IR InfraredISO International Organization for Standardization IV Intravenous JPJapanese Pharmacopoeia KF Karl Fischer Titration LC LiquidChromatography LC-MS Liquid Chromatography with Mass Spectrometry LVLeft Ventricular LVdP/dt Left Ventricular Contractility mcg Micrograms,μg or ug MDI Metered Dose Inhaler MeOH Methanol MLWHF Minnesota LivingWith Heart Failure Questionaire MMAD Mass Median Aerodynamic DiameterMTD Maximum Tolerated Dose PAPm Mean Pulmonary Arterial Pressure NDA NewDrug Application NF National Formulary NGI Next Generation Impactor ™NMR Nuclear Magnetic Resonance NMT Not More Than NO Nitric Oxide NOAELNo Observed Adverse Effect Level NRF Normal Renal Function NT Not TestedNT-proBNP N-Terminal of the Prohormone Brain Natriuretic Peptide NYHANew York Heart Association OHSAS Occupational Health and Safety AdvisoryServices OPP Oriented Polypropylene PAH Pulmonary Arterial HypertensionPAP Pulmonary Arterial Pressure PCW Pulmonary Capillary Wedge pressurePD Pharmacodynamics PDE5 Phosphodiesterase Type 5 Inhibitors PEPolyethylene PET Polyethylene Terephthalate PGI₂ Prostaglandin I2(Prostacyclin) PH Pulmonary Hypertension PK Pharmacokinetics PPM PartsPer Million PRINT Particle Replication In Nonwetting Templates PTFEPolytetrafluoroethylene PVR Pulmonary Vascular Resistance QID Quarter inDie (Four Times Daily) (Q)SAR Quantitative Structure-ActivityRelationship QTc Corrected QT Interval RH Relative Humidity RLDReference Listed Drug SAC Single Actuation Content SAE Serious AdverseEvent SAP Systemic Arterial Pressure SC Subcutaneous SEM Standard Errorof the Mean SOP Standard Operating Procedure SVR Systemic VascularResistance t½ Half-life TBD To Be Determined TK Toxicokinetics Tmax Timeof Maximal Concentration TMB acid Trimethylbenzoic Acid TMB-AldTrimethylbenzaldehyde TMP Trimethylbenzoyl Diphenylphosphine OxideTRIUMPH Treprostinil Sodium Inhalation Used in the Management ofPulmonary Arterial Hypertension (clinical trial) TTC Threshold forToxicological Concern IUPAC International Union of Pure and AppliedChemistry μg or ug Micrograms or mcg US United States USP United StatesPharmacopeia WHO World Health Organization WRS Wilcoxon Rank Sum Test WTWeight XRD X-ray Diffractometer

Product Nomenclature and Reference Table Term Designation ExemplaryEmbodiment Drug Substance DS Treprostinil, supplied as treprostinil orsodium for manufacture of the LIQ861 treprostinil drugproduct-intermediate Drug Product-Intermediate DP-intermediate Drypowder particles of precise size and or shape containing an integratedmatrix of LIQ861 DP-intermediate treprostinil and excipients that isproduced using Liquidia's PRINT ® Technology manufacturing process (bulkdry powder prior to capsule filling) Placebo Drug Product- PlaceboDP-intermediate Identical formulation as the DP- IntermediateIntermediate, but treprostinil is replaced with an equal mass oftrehalose Inhalation Powder Drug DP or LIQ861 DP-intermediate ProductLIQ861 Drug Product filled into Size 3 HPMC capsules for oral or LIQ861inhalation, but prior to integration with Inhalation Device Placebo DrugProduct Placebo Placebo DP-intermediate filled into Size 3 hydroxypropylmethylcellulose (HPMC) capsules, but prior to integration with theInhalation Device Drug Product Strength Treprostinil in LIQ861 Amount oftreprostinil in drug product or Dose Packaged Drug Product None DrugProduct in the Primary Packaging Inhalation Device Device Device that isused to deliver the Drug Product Premetered Dry Powder DPI Drug Productintegrated with the Inhaler Inhalation Device; i.e., the final productfor patient use

We claim:
 1. A method for treating a patient having pulmonary arterialhypertension, comprising: administration of a dry powder compositioncomprising from about 100 micrograms to about 300 microgramstreprostinil or a pharmaceutically acceptable salt thereof to a patientby inhalation using a dry powder inhaler over one to four breaths totreat pulmonary arterial hypertension.
 2. The method of claim 1, whereinthe patient receives greater than about 50 micrograms treprostinil perbreath.
 3. The method of claim 1, wherein the dry powder compositioncomprises less than or equal to about 30 mg of dry powder per breath. 4.The method of claim 1, wherein the dry powder composition comprisesmolded dry particles comprising a non-reducing sugar, a wetting agent, ahydrophobicity modifying agent, a pH modifying agent and a buffer. 5.The method of claim 1, wherein the dry powder composition comprisesmolded dry particles comprising by percent solids about 0.581 percenttreprostinil sodium, about 92.32 percent trehalose, about 2.19 percentpolysorbate 80, about 4.39 percent L-leucine, about 0.26 percent sodiumcitrate, and about 0.25 percent sodium chloride.
 6. The method of claim1, wherein the patient inhales the dry powder composition four times aday.
 7. The method of claim 6, wherein the patient inhales a fifth doseof the dry powder composition per day.
 8. The method of claim 6, whereinthe patient inhales between 100 micrograms and 300 microgramstreprostinil or a pharmaceutically acceptable salt thereof through fourbreaths over a day.
 9. The method of claim 6, wherein the patientinhales between 100 micrograms and 600 micrograms treprostinil or apharmaceutically acceptable salt thereof through four to sixteen breathsper day.
 10. A method of treating a patient having pulmonary arterialhypertension, comprising: providing a patient a dry powder inhaler;providing the patient at least one capsule for use in the dry powderinhaler, wherein the at least one capsule contains a dry powdercomposition comprising at least 100 micrograms of treprostinil, anon-reducing sugar, a wetting agent, a hydrophobicity modifying agent, apH modifying agent and a buffer; and instructing the patient to utilizethe dry powder inhaler to inhale the dry powder composition from eachcapsule in one to two breaths.
 11. The method of claim 10, wherein thecapsule comprises at least 150 micrograms of treprostinil.
 12. A methodof treating a patient having pulmonary arterial hypertension,comprising: dosing the patient having pulmonary arterial hypertensionwith a dry powder dose of treprostinil, wherein the dose of treprostinilis from about 100 micrograms to about 300 micrograms and delivered overone to four breaths, wherein the dry powder dose is configured intoparticles, wherein each particle comprises the treprostinil, anon-reducing sugar, a wetting agent, a hydrophobicity modifying agent, apH modifying agent and a buffer.
 13. A method for treating a patienthaving pulmonary arterial hypertension, comprising: administering a drypowder composition comprising between about 400 micrograms treprostiniland about 1200 micrograms treprostinil to a patient over a day throughfour to sixteen breaths to treat pulmonary arterial hypertension,wherein the dry powder composition is configured as uniform particles.14. The method of claim 13, wherein the about 400 microgramstreprostinil is inhaled by the patient in eight breaths or less per day.15. The method of claim 13, wherein about 800 micrograms treprostinil isinhaled by the patient in eight breaths or less per day.
 16. The methodof claim 13, wherein about 1200 micrograms treprostinil is inhaled bythe patient in sixteen breaths or less per day.
 17. The method of claim1, wherein the dry powder composition induces fewer side effects than anebulized dose of 54 micrograms of treprostinil.
 18. The method of claim10, wherein the dry powder composition induces fewer side effects than anebulized dose of 54 micrograms of treprostinil.
 19. The method of claim13, wherein the dry powder dose of treprostinil induces fewer sideeffects than a nebulized dose of 54 micrograms of treprostinil.